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suppression of host gene expression by nsp1 proteins of group 2 bat coronaviruses.nsp1 protein of severe acute respiratory syndrome coronavirus (sars-cov), a group 2b cov, suppresses host gene expression by promoting host mrna degradation and translation inhibition. the present study analyzed the activities of nsp1 proteins from the group 2 bat cov strains rm1, 133, and hku9-1, belonging to groups 2b, 2c, and 2d, respectively. the host mrna degradation and translational suppression activities of nsp1 of sars-cov and rm1 nsp1 were similar and stronger than the activities of th ...200919264783
drug targets in severe acute respiratory syndrome (sars) virus and other coronavirus infections.coronaviruses are important human and animal pathogens of the order nidovirales. several new members were discovered following the emergence of sars-cov in human populations, including two human coronaviruses and several animal coronaviruses. they cause respiratory and gastrointestinal illnesses and have been found in the brains of patients with multiple sclerosis. the high mortality of sars, the identification of a natural reservoir, and the well-founded fear of provoking antibody-enhanced dise ...200919275708
timely identification of optimal control strategies for emerging infectious diseases.health authorities must rely on quarantine, isolation, and other non-pharmaceutical interventions to contain outbreaks of newly emerging human diseases.200919289133
neutralizing antibody against severe acute respiratory syndrome (sars)-coronavirus spike is highly effective for the protection of mice in the murine sars model.we evaluated the efficacy of three sars vaccine candidates in a murine sars model utilizing low-virulence pp and sars-cov coinfection. vaccinated mice were protected from severe respiratory disease in parallel with a low virus titer in the lungs and a high neutralizing antibody titer in the plasma. importantly, the administration of spike protein-specific neutralizing monoclonal antibody protected mice from the disease, indicating that the neutralization is sufficient for protection. moreover, a ...200919291090
differential virological and immunological outcome of severe acute respiratory syndrome coronavirus infection in susceptible and resistant transgenic mice expressing human angiotensin-converting enzyme 2.we previously reported that transgenic (tg) mice expressing human angiotensin-converting enzyme 2 (hace2), the receptor for severe acute respiratory syndrome coronavirus (sars-cov), were highly susceptible to sars-cov infection, which resulted in the development of disease of various severity and even death in some lineages. in this study, we further characterized and compared the pathogeneses of sars-cov infection in two of the most stable tg lineages, ac70 and ac22, representing those suscepti ...200919297479
biochemical characterization of arterivirus nonstructural protein 11 reveals the nidovirus-wide conservation of a replicative endoribonuclease.nidoviruses (arteriviruses, coronaviruses, and roniviruses) are a phylogenetically compact but diverse group of positive-strand rna viruses that includes important human and animal pathogens. nidovirus rna synthesis is mediated by a cytoplasmic membrane-associated replication/transcription complex that includes up to 16 viral nonstructural proteins (nsps), which carry common enzymatic activities, like the viral rna polymerase, but also unusual and poorly understood rna-processing functions. of t ...200919297500
the 8ab protein of sars-cov is a luminal er membrane-associated protein and induces the activation of atf6.the 8ab protein of sars-cov is a group-specific accessory protein, which is lost when the virus was transmitted from animals to humans due to a 29-nucleotide deletion in the orf8ab region. here we found that 8ab protein is associated with er membrane at luminal surface. 8ab protein was found to up-regulate the synthesis of endogenous er-resident chaperons involved in protein folding through the activation of the transcription factor atf6, while it showed no effect on the chop induction and xbp1 ...200919304306
[prokaryotic expression of s2 extracellular domain of sars coronavirus spike protein and its fusion with hela cell membrane].to construct the expression plasmid of s2 extracellular domain (s2ed) of sars-coronavirus (sars- cov) spike protein (s protein) and enhanced green fluorescent protein (egfp) to obtain the fusion protein expressed in prokaryotic cells.200919304506
c-terminal domain of sars-cov main protease can form a 3d domain-swapped dimer.sars coronavirus main protease (m(pro)) plays an essential role in the extensive proteolytic processing of the viral polyproteins (pp1a and pp1ab), and it is an important target for anti-sars drug development. we have reported that both the m(pro) c-terminal domain alone (m(pro)-c) and the n-finger deletion mutant of m(pro) (m(pro)-delta7) exist as a stable dimer and a stable monomer (zhong et al., j virol 2008; 82:4227-4234). here, we report structures of both m(pro)-c monomer and dimer. the st ...200919319935
activation of the sars coronavirus spike protein via sequential proteolytic cleavage at two distinct sites.the coronavirus spike protein (s) plays a key role in the early steps of viral infection, with the s1 domain responsible for receptor binding and the s2 domain mediating membrane fusion. in some cases, the s protein is proteolytically cleaved at the s1-s2 boundary. in the case of the severe acute respiratory syndrome coronavirus (sars-cov), it has been shown that virus entry requires the endosomal protease cathepsin l; however, it was also found that infection of sars-cov could be strongly induc ...200919321428
expression and membrane integration of sars-cov e protein and its interaction with m protein.the severe acute respiratory syndrome (sars)-cov e gene fragment was cloned and expressed as a recombinant protein fused with a myc tag at the n-terminus in vitro and in vero e6 cells. similar to other n-glycosylated proteins, the glycosylation of sars-cov e protein occurred co-translationally in the presence of microsomes. the sars-cov e protein is predicted to be a double-spanning membrane protein lacking a conventional signal peptide. both of the transmembrane regions (a.a. 11-33 and 37-59) a ...200919322648
structural insights into immune recognition of the severe acute respiratory syndrome coronavirus s protein receptor binding domain.the spike (s) protein of the severe acute respiratory syndrome coronavirus (sars-cov) is responsible for host cell attachment and fusion of the viral and host cell membranes. within s the receptor binding domain (rbd) mediates the interaction with angiotensin-converting enzyme 2 (ace2), the sars-cov host cell receptor. both s and the rbd are highly immunogenic and both have been found to elicit neutralizing antibodies. reported here is the x-ray crystal structure of the rbd in complex with the f ...200919324051
apobec3g cytidine deaminase association with coronavirus nucleocapsid protein.we previously reported that replacing hiv-1 nucleocapsid (nc) domain with sars-cov nucleocapsid (n) residues 2-213, 215-421, or 234-421 results in efficient virus-like particle (vlp) production at a level comparable to that of wild-type hiv-1. in this study we demonstrate that these chimeras are capable of packaging large amounts of human apobec3g (ha3g), and that an hiv-1 gag chimera containing the carboxyl-terminal half of human coronavirus 229e (hcov-229e) n as a substitute for nc is capable ...200919345973
new developments for the design, synthesis and biological evaluation of potent sars-cov 3cl(pro) inhibitors.a series of trifluoromethyl, benzothiazolyl or thiazolyl ketone-containing peptidic compounds as sars-cov 3cl protease inhibitors were developed and their potency was evaluated by in vitro protease inhibitory assays. three candidates had encouraging results for the development of new anti-sars compounds.200919362479
translational control of the subgenomic rnas of severe acute respiratory syndrome coronavirus.the 3'-one-third of the severe acute respiratory syndrome coronavirus (sars-cov) genome contains genes for four essential structural proteins and eight virus-specific genes. the expression of this genomic information of sars-cov involves synthesis of a nested set of subgenomic rnas (sgrnas). in this study, we showed that the translational levels of 10 sars-cov sgrnas including the two low-abundance sgrnas 2-1 and 3-1 varied considerably in translation reporter assays. we also demonstrated that t ...200919363699
mrna display design of fibronectin-based intrabodies that detect and inhibit severe acute respiratory syndrome coronavirus nucleocapsid protein.the nucleocapsid (n) protein of severe acute respiratory syndrome (sars) coronavirus plays important roles in both viral replication and modulation of host cell processes. new ligands that target the n protein may thus provide tools to track the protein inside cells, detect interaction hot spots on the protein surface, and discover sites that could be used to develop new anti-sars therapies. using mrna display selection and directed evolution, we designed novel antibody-like protein affinity rea ...200919364769
severe acute respiratory syndrome coronavirus papain-like protease ubiquitin-like domain and catalytic domain regulate antagonism of irf3 and nf-kappab signaling.the outcome of a viral infection is regulated in part by the complex coordination of viral and host interactions that compete for the control and optimization of virus replication. severe acute respiratory syndrome coronavirus (sars-cov) intimately engages and regulates the host innate immune responses during infection. using a novel interferon (ifn) antagonism screen, we show that the sars-cov proteome contains several replicase, structural, and accessory proteins that antagonize the ifn pathwa ...200919369340
the sr-rich motif in sars-cov nucleocapsid protein is important for virus replication.the multimerization/self-interaction of viral proteins is an important step in the process of viral assembly and maturation. our previous study indicated that the severe acute respiratory syndrome-associated coronavirus (sars-cov) nucleocapsid protein forms self-multimers through a serine-arginine (sr)-rich motif (ssrsssrsrgnsr) by using a mammalian two-hybrid system. to determine the biological relevance of this motif, we constructed a sars-cov reverse genetic construct by using a bacterial art ...200919370068
thiopurine analogue inhibitors of severe acute respiratory syndrome-coronavirus papain-like protease, a deubiquitinating and deisgylating enzyme.in the search for effective therapeutics against severe acute respiratory syndrome (sars), 6-mercaptopurine (6mp) and 6-thioguanine (6tg) were found to be specific inhibitors for the sars-coronavirus (cov) papain-like protease (plpro), a cysteine protease with deubiquitinating and deisgylating activity. 6mp and 6tg have long been used in cancer chemotherapy for treatment of acute lymphoblastic or myeloblastic leukaemia. development and optimization of 6mp and 6tg will not only be important for a ...200919374142
use of gfp to investigate expression of plant-derived vaccines.plants are low-cost bioreactors for the production of various biopharmaceuticals including oral vaccines. plant-derived oral vaccines are potentially useful in combating viral infections involving mucosal immunity. transgenic plants have been generated to successfully produce mucosal vaccines against cholera, hepatitis b, foot-and-mouth disease, and norwalk virus. as a first step toward the generation of oral vaccines against the severe acute respiratory syndrome coronavirus (sars-cov), we have ...200919378126
severe acute respiratory syndrome coronavirus m protein inhibits type i interferon production by impeding the formation of traf3.tank.tbk1/ikkepsilon complex.severe acute respiratory syndrome (sars) coronavirus is highly pathogenic in humans and evades innate immunity at multiple levels. it has evolved various strategies to counteract the production and action of type i interferons, which mobilize the front-line defense against viral infection. in this study we demonstrate that sars coronavirus m protein inhibits gene transcription of type i interferons. m protein potently antagonizes the activation of interferon-stimulated response element-dependent ...200919380580
incubation periods of acute respiratory viral infections: a systematic review.knowledge of the incubation period is essential in the investigation and control of infectious disease, but statements of incubation period are often poorly referenced, inconsistent, or based on limited data. in a systematic review of the literature on nine respiratory viral infections of public-health importance, we identified 436 articles with statements of incubation period and 38 with data for pooled analysis. we fitted a log-normal distribution to pooled data and found the median incubation ...200919393959
severe acute respiratory syndrome coronavirus accessory protein 9b is a virion-associated protein.eight accessory proteins have been identified in severe acute respiratory syndrome-associated coronavirus (sars-cov). they are believed to play roles in the viral life cycle and may contribute to the pathogenesis and virulence. orf9b as one of these accessory proteins is located in subgenomic mrna9 and encodes a 98 amino acid protein. however, whether 9b protein is a structural component of sars-cov particles remains unknown. in this study, we demonstrate that 9b protein is translated from bicis ...200919394665
determining sars sub-clinical infection: a longitudinal seroepidemiological study in recovered sars patients and controls after an outbreak in a general hospital.a cohort of 67 confirmed sars patients were prospectively followed for 16 months and were compared with a control population. serum samples taken at various times were tested for igg and igm; dynamic serological changes in these antibodies were described. the positive responses of igm and igg antibodies in sera against sars virus from the first week to the sixth week after onset of the illness in patients with sars were measured. the elisa test of igg antibody was negative in 200 community contr ...200919396666
the ion channel activity of the sars-coronavirus 3a protein is linked to its pro-apoptotic function.the severe acute respiratory syndrome-coronavirus (sars-cov) caused an outbreak of atypical pneumonia in 2003. the sars-cov viral genome encodes several proteins which have no homology to proteins in any other coronaviruses, and a number of these proteins have been implicated in viral cytopathies. one such protein is 3a, which is also known as x1, orf3 and u274. 3a expression is detected in both sars-cov infected cultured cells and patients. among the different functions identified, 3a is a capa ...200919398035
expression and characterization of recombinant s2 subunit of sars-coronavirus s fusion protein. 200919400136
molecular determinants for subcellular localization of the severe acute respiratory syndrome coronavirus open reading frame 3b protein.viruses such as hepatitis c and the severe acute respiratory syndrome coronavirus (sars-cov) encode proteins that are distributed between mitochondria and the nucleus, but little is known about the factors that control partitioning between these sites. sars-cov encodes a unique accessory gene called open reading frame (orf) 3b that, like other unique accessory genes in sars-cov, likely contributes to viral pathogenicity. the orf 3b protein is 154 amino acids and is predicted to express from the ...200919403678
mavs-mediated apoptosis and its inhibition by viral proteins.host responses to viral infection include both immune activation and programmed cell death. the mitochondrial antiviral signaling adaptor, mavs (ips-1, visa or cardif) is critical for host defenses to viral infection by inducing type-1 interferons (ifn-i), however its role in virus-induced apoptotic responses has not been elucidated.200919404494
lack of association between polymorphisms of masp2 and susceptibility to sars coronavirus infection.the pathogenesis of severe acute respiratory disease syndrome (sars) is not fully understood. one case-control study has reported an association between susceptibility to sars and mannan-binding lectin (mbl) in china. as the downstream protein of mbl, variants of the mbl-associated serine protease-2 (masp2) gene may be associated with sars coronavirus (sars-cov) infection in the same population.200919405982
expression, crystallization and preliminary crystallographic study of human coronavirus hku1 nonstructural protein 9.human coronavirus hku1 (hcov-hku1) belongs to coronavirus group ii and encodes 16 nonstructural proteins (nsps) which mediate genome replication and transcription. among these nsps, nsp9 has been shown to possess single-stranded dna/rna-binding properties. the gene that encodes hcov-hku1 nsp9 was cloned and expressed in escherichia coli and the protein was subjected to crystallization trials. the crystals diffracted to 2.7 a resolution and belonged to space group p2(1)2(1)2, with unit-cell param ...200919407394
towards construction of viral vectors based on avian coronavirus infectious bronchitis virus for gene delivery and vaccine development.manipulation of the coronavirus genome to accommodate and express foreign genes is an attractive approach for gene delivery and vaccine development. by using an infectious cloning system developed recently for the avian coronavirus infectious bronchitis virus (ibv), the enhanced green fluorescent protein (egfp) gene, the firefly luciferase gene and several host and viral genes (eif3f, sars orf6, dengue virus 1 core protein gene) were inserted into various positions of the ibv genome, and the eff ...200919409420
two adjacent mutations on the dimer interface of sars coronavirus 3c-like protease cause different conformational changes in crystal structure.the 3c-like protease of sars coronavirus (sars-cov 3cl(pro)) is vital for sars-cov replication and is a promising drug target. it has been extensively proved that only the dimeric enzyme is active. here we discovered that two adjacent mutations (ser139_ala and phe140_ala) on the dimer interface resulted in completely different crystal structures of the enzyme, demonstrating the distinct roles of these two residues in maintaining the active conformation of sars-cov 3cl(pro). s139a is a monomer th ...200919409595
ectodomain shedding of angiotensin converting enzyme 2 in human airway epithelia.angiotensin-converting enzyme 2 (ace2) is a terminal carboxypeptidase and the receptor for the sars and nl63 coronaviruses (cov). loss of ace2 function is implicated in severe acute respiratory syndrome (sars) pathogenesis, but little is known about ace2 biogenesis and activity in the airways. we report that ace2 is shed from human airway epithelia, a site of sars-cov infection. the regulation of ace2 release was investigated in polarized human airway epithelia. constitutive generation of solubl ...200919411314
interaction of a peptide corresponding to the loop domain of the s2 sars-cov virus protein with model membranes.the severe acute respiratory syndrome coronavirus (sars-cov) envelope spike (s) glycoprotein is responsible for the fusion between the membranes of the virus and the target cell. in the case of the s2 domain of protein s, it has been found a highly hydrophobic and interfacial domain flanked by the heptad repeat 1 and 2 regions; significantly, different peptides pertaining to this domain have shown a significant leakage effect and an important plaque formation inhibition, which, similarly to hiv- ...200919412834
identification of in vivo-interacting domains of the murine coronavirus nucleocapsid protein.the coronavirus nucleocapsid protein (n), together with the large, positive-strand rna viral genome, forms a helically symmetric nucleocapsid. this ribonucleoprotein structure becomes packaged into virions through association with the carboxy-terminal endodomain of the membrane protein (m), which is the principal constituent of the virion envelope. previous work with the prototype coronavirus mouse hepatitis virus (mhv) has shown that a major determinant of the n-m interaction maps to the carbox ...200919420077
early upregulation of acute respiratory distress syndrome-associated cytokines promotes lethal disease in an aged-mouse model of severe acute respiratory syndrome coronavirus infection.several respiratory viruses, including influenza virus and severe acute respiratory syndrome coronavirus (sars-cov), produce more severe disease in the elderly, yet the molecular mechanisms governing age-related susceptibility remain poorly studied. advanced age was significantly associated with increased sars-related deaths, primarily due to the onset of early- and late-stage acute respiratory distress syndrome (ards) and pulmonary fibrosis. infection of aged, but not young, mice with recombina ...200919420084
development of a chimeric dna-rna hammerhead ribozyme targeting sars virus.severe acute respiratory syndrome (sars) is a severe pulmonary infectious disease caused by a novel coronavirus. to develop an effective and specific medicine targeting the sars-coronavirus (cov), a chimeric dna-rna hammerhead ribozyme was designed and synthesized using a sequence homologous with the mouse hepatitis virus (mhv).200919420961
label-free, electrical detection of the sars virus n-protein with nanowire biosensors utilizing antibody mimics as capture probes.antibody mimic proteins (amps) are polypeptides that bind to their target analytes with high affinity and specificity, just like conventional antibodies, but are much smaller in size (2-5 nm, less than 10 kda). in this report, we describe the first application of amp in the field of nanobiosensors. in(2)o(3) nanowire based biosensors have been configured with an amp (fibronectin, fn) to detect nucleocapsid (n) protein, a biomarker for severe acute respiratory syndrome (sars). using these devices ...200919422193
antigenicity and immunogenicity of sars-cov s protein receptor-binding domain stably expressed in cho cells.the receptor-binding domain (rbd) of sars coronavirus (sars-cov) spike (s) protein contains multiple conformation-dependent epitopes that induce neutralizing antibody responses. here we used cho-k1 cells to establish a cell line for stable expression of a 193-mer (residues 318-510) rbd (rbd193-cho) and determined its antigenicity and immunogenicity. we found that rbd193-cho reacted strongly with a panel of six monoclonal antibodies recognizing various conformational and linear epitopes in rbd, s ...200919422787
severe acute respiratory syndrome coronavirus nucleocapsid protein does not modulate transcription of the human fgl2 gene.among the structural and nonstructural proteins of severe acute respiratory syndrome coronavirus (sars-cov), the nucleocapsid (n) protein plays pivotal roles in the biology and pathogenesis of viral infection. n protein is thought to dysregulate cell signalling and the transcription of cellular genes, including fgl2, which encodes a prothrombinase implicated in vascular thrombosis, fibrin deposition and pneumocyte necrosis. here, we showed that n protein expressed in cultured human cells was pre ...200919423547
procyanidins and butanol extract of cinnamomi cortex inhibit sars-cov infection.we found that the butanol fraction of cinnamomi cortex (cc/fr.2) showed moderate inhibitory activity in wild-type severe acute respiratory syndrome coronavirus (wtsars-cov) and hiv/sars-cov s pseudovirus infections. the inhibition on pseudovirus was also seen in cells pretreated with the cc and cc/fr.2 (ic(50s), 283.4+/-16.3 and 149.5+/-13.5 microg/ml, respectively), however the highest activities on wtsars-cov were observed when the viruses were treated by the extracts before challenging (ic(50 ...200919428598
toward better clinical data in emerging infectious diseases. 200919435434
the sars-unique domain (sud) of sars coronavirus contains two macrodomains that bind g-quadruplexes.since the outbreak of severe acute respiratory syndrome (sars) in 2003, the three-dimensional structures of several of the replicase/transcriptase components of sars coronavirus (sars-cov), the non-structural proteins (nsps), have been determined. however, within the large nsp3 (1922 amino-acid residues), the structure and function of the so-called sars-unique domain (sud) have remained elusive. sud occurs only in sars-cov and the highly related viruses found in certain bats, but is absent from ...200919436709
elevated plasma surfactant protein d (sp-d) levels and a direct correlation with anti-severe acute respiratory syndrome coronavirus-specific igg antibody in sars patients.pulmonary sp-d is a defence lectin promoting clearance of viral infections. sp-d is recognized to bind the s protein of sars-cov and enhance phagocytosis. moreover, systemic sp-d is widely used as a biomarker of alveolar integrity. we investigated the relation between plasma sp-d, sars-type pneumonia and the sars-specific igg response. sixteen patients with sars, 19 patients with community-acquired pneumonia (cap) (streptococcus pneumonia) and 16 healthy control subjects were enrolled in the stu ...200919439011
characterization of a highly conserved domain within the severe acute respiratory syndrome coronavirus spike protein s2 domain with characteristics of a viral fusion peptide.many viral fusion proteins are primed by proteolytic cleavage near their fusion peptides. while the coronavirus (cov) spike (s) protein is known to be cleaved at the s1/s2 boundary, this cleavage site is not closely linked to a fusion peptide. however, a second cleavage site has been identified in the severe acute respiratory syndrome cov (sars-cov) s2 domain (r797). here, we investigated whether this internal cleavage of s2 exposes a viral fusion peptide. we show that the residues immediately c ...200919439480
therapies for coronaviruses. part 2: inhibitors of intracellular life cycle.severe acute respiratory syndrome (sars) coronavirus emerged from an animal reservoir in 2002 and has the potential to reemerge, as shown by the occurrence of non-laboratory-associated new cases in the winter of 2003. in the absence of a vaccine, broad spectrum anticoronaviral medications are needed.200919441924
review of new and newly discovered respiratory tract viruses in children.respiratory tract viral infection continues to be among the most common reasons for emergency department visits and hospitalization of children, particularly infants younger than 1 year, in the united states. throughout the years, clinicians have considered respiratory syncytial virus followed by influenza as the most common pathogens responsible. over the past decade, new viruses have been discovered through both more specific testing and the finding of new agents causing infection. this includ ...200919444037
therapies for coronaviruses. part i of ii -- viral entry inhibitors.severe acute respiratory syndrome (sars) coronavirus emerged fleetingly in the winter of 2002 and again in the winter of 2003, resulting in the infection of ~8,000 people and the death of ~800. the identification of the putative natural reservoir suggests that a re-emergence is possible. the functions of many coronaviral proteins have now been elucidated, resulting in many novel approaches to therapy.200919449500
sars-coronavirus modulation of myocardial ace2 expression and inflammation in patients with sars.angiotensin converting enzyme 2 (ace2), a monocarboxylase that degrades angiotensin ii to angiotensin 1-7, is also the functional receptor for severe acute respiratory syndrome (sars) coronavirus (sars-cov) and is highly expressed in the lungs and heart. patients with sars also suffered from cardiac disease including arrhythmias, sudden cardiac death, and systolic and diastolic dysfunction.200919453650
rapid pulmonary fibrosis induced by acute lung injury via a lipopolysaccharide three-hit regimen.based on the common characteristic of severe acute respiratory syndrome (sars) and highly pathogenic avian influenza and the mechanism of inflammation and fibrosis, it is speculated that there should exist a fundamental pathological rule that severe acute lung injury (ali)-induced rapid pulmonary fibrosis is caused by various etiological factors, such as sars coronavirus, h5n1-virus, or other unknown factors, and also by lipopolysaccharide (lps), the most common etiological factor. the investiga ...200919474208
characterization of cytotoxic t-lymphocyte epitopes and immune responses to sars coronavirus spike dna vaccine expressing the rgd-integrin-binding motif.integrins are critical for initiating t-cell activation events. the integrin-binding motif arg-gly-asp (rgd) was incorporated into the pcdna 3.1 mammalian expression vector expressing the codon-optimized extracellular domain of sars coronavirus (sars-cov) spike protein, and tested by immunizing c57bl/6 mice. significant cell-mediated immune responses were characterized by cytotoxic t-lymphocyte (51)cr release assay and interferon-gamma secretion elispot assay against rma-s target cells presentin ...200919475608
synthetic peptides coupled to the surface of liposomes effectively induce sars coronavirus-specific cytotoxic t lymphocytes and viral clearance in hla-a*0201 transgenic mice.we investigated whether the surface-linked liposomal peptide was applicable to a vaccine based on cytotoxic t lymphocytes (ctls) against severe acute respiratory syndrome (sars) coronavirus (sars-cov). we first identified four hla-a*0201-restricted ctl epitopes derived from sars-cov using hla-a*0201 transgenic mice and recombinant adenovirus expressing predicted epitopes. these peptides were coupled to the surface of liposomes, and inoculated into mice. two of the liposomal peptides were effecti ...200919490987
bats and emerging zoonoses: henipaviruses and sars.nearly 75% of all emerging infectious diseases (eids) that impact or threaten human health are zoonotic. the majority have spilled from wildlife reservoirs, either directly to humans or via domestic animals. the emergence of many can be attributed to predisposing factors such as global travel, trade, agricultural expansion, deforestation/habitat fragmentation, and urbanization; such factors increase the interface and/or the rate of contact between human, domestic animal, and wildlife populations ...200919497090
management guidelines for obstetric patients and neonates born to mothers with suspected or probable severe acute respiratory syndrome (sars).this document summarizes the limited experience of sars in pregnancy and suggests guidelines for management.200919497157
toll-like receptors, chemokine receptors and death receptor ligands responses in sars coronavirus infected human monocyte derived dendritic cells.the sars outbreak in 2003 provides a unique opportunity for the study of human responses to a novel virus. we have previously reported that dendritic cells (dcs) might be involved in the immune escape mechanisms for sars-cov. in this study, we focussed on the gene expression of toll-like receptors (tlrs), chemokine receptors (ccrs) and death receptor ligands in sars-cov infected dcs. we also compared adult and cord blood (cb) dcs to find a possible explanation for the age-dependent severity of s ...200919505311
antiviral activity of chloroquine against human coronavirus oc43 infection in newborn mice.until recently, human coronaviruses (hcovs), such as hcov strain oc43 (hcov-oc43), were mainly known to cause 15 to 30% of mild upper respiratory tract infections. in recent years, the identification of new hcovs, including severe acute respiratory syndrome coronavirus, revealed that hcovs can be highly pathogenic and can cause more severe upper and lower respiratory tract infections, including bronchiolitis and pneumonia. to date, no specific antiviral drugs to prevent or treat hcov infections ...200919506054
development of interfering rna agents to inhibit sars-associated coronavirus infection and replication. 200919509435
construction of plasmids expressing sars-cov encoding proteins and their effects on transcription of hfgl2 prothrombinase.sars coronavirus (sars-cov) is the etiologic agent of severe acute respiratory syndrome. the aim of this study was to construct sars-cov membrane (m), nucleocapsid (n) and spike 2 (s2) gene eukaryotic expression plasmids, and identify their expression in vitro. gene fragments encoding n protein, m protein and s2 protein of sars-cov were amplified by pcr using cdna obtained from lung samples of sars patients as template, and subcloned into pcdna3.1 vector to form eukaryotic expression plasmids. s ...200919513614
multiplex pcr tests sentinel the appearance of pandemic influenza viruses including h1n1 swine influenza.since the turn of the century seven new respiratory viruses have infected man and two of these have resulted in worldwide epidemics. both sars coronavirus which quickly spread to 29 countries in february 2003 and h1n1 swine influenza that recently spread from mexico to 30 countries in three weeks represent major pandemic threats for mankind. diagnostic assays are required to detect novel influenza strains with pandemic potential.200919515609
boosted expression of the sars-cov nucleocapsid protein in tobacco and its immunogenicity in mice.vaccines produced in plant systems are safe and economical; however, the extensive application of plant-based vaccines is mainly hindered by low expression levels of heterologous proteins in plant systems. here, we demonstrated that the post-transcriptional gene silencing suppressor p19 protein from tomato bushy stunt virus substantially enhanced the transient expression of recombinant sars-cov nucleocapsid (rn) protein in nicotiana benthamiana. the rn protein in the agrobacteria-infiltrated pla ...200919523911
characterization of sars-cov-specific memory t cells from recovered individuals 4 years after infection.sars-cov infection of human results in antigen-specific cellular and humoral immune responses. however, it is critical to determine whether sars-cov-specific memory t cells can persist for long periods of time. in this study, we analyzed the cellular immune response from 21 sars-recovered individuals who had been diagnosed with sars in 2003 by using elisa, cba, elispot and multiparameter flow cytometry assays. our results demonstrated that low levels of specific memory t cell responses to sars-c ...200919526193
studies on membrane topology, n-glycosylation and functionality of sars-cov membrane protein.the glycosylated membrane protein m of the severe acute respiratory syndrome associated coronavirus (sars-cov) is the main structural component of the virion and mediates assembly and budding of viral particles. the membrane topology of sars-cov m and the functional significance of its n-glycosylation are not completely understood as is its interaction with the surface glycoprotein s. using biochemical and immunofluorescence analyses we found that m consists of a short glycosylated n-terminal ec ...200919534833
sars vaccines: where are we?in this review, the current state of vaccine development against human severe acute respiratory syndrome (sars) coronavirus, focusing on recently published data is assessed. we discuss which strategies have been assessed immunologically and which have been evaluated in sars coronavirus challenge models. we discuss inactivated vaccines, virally and bacterially vectored vaccines, recombinant protein and dna vaccines, as well as the use of attenuated vaccines. data regarding the correlates of prote ...200919538115
a computational analysis of sars cysteine proteinase-octapeptide substrate interaction: implication for structure and active site binding mechanism.sars coronavirus main proteinase (sars covmpro) is an important enzyme for the replication of severe acute respiratory syndrome virus. the active site region of sars covmpro is divided into 8 subsites. understanding the binding mode of sars covmpro with a specific substrate is useful and contributes to structural-based drug design. the purpose of this research is to investigate the binding mode between the sars covmpro and two octapeptides, especially in the region of the s3 subsite, through a m ...200919208150
functional screen reveals sars coronavirus nonstructural protein nsp14 as a novel cap n7 methyltransferase.the n7-methylguanosine (m7g) cap is the defining structural feature of eukaryotic mrnas. most eukaryotic viruses that replicate in the cytoplasm, including coronaviruses, have evolved strategies to cap their rnas. in this report, we used a yeast genetic system to functionally screen for the cap-forming enzymes encoded by severe acute respiratory syndrome (sars) coronavirus and identified the nonstructural protein (nsp) 14 of sars coronavirus as a (guanine-n7)-methyltransferase (n7-mtase) in vivo ...200919208801
sars-cov spike proteins expressed by the vaccinia virus tiantan strain: secreted sq protein induces robust neutralization antibody in mice.the spike (s) glycoprotein of severe acute respiratory syndrome coronavirus (sars-cov) is a major target in the development of diagnostic assays and vaccines, but its antigenic and immunogenic properties remain unclear. seven sars-cov spike proteins (s, sq, s1, rbd, s2, s2q, and cx) were generated using the modified vaccinia virus (tiantan strain) as a vector, and their antigenicity and immunogenicity were evaluated. the secreted sq protein in which the transmembrane domain was deleted, as well ...200919210229
potent human monoclonal antibodies against sars cov, nipah and hendra viruses.background: recently, several potently neutralizing fully human monoclonal antibodies (hmabs) targeting the severe acute respiratory syndrome-associated coronavirus (sars cov) s glycoprotein, and the g glycoprotein of the paramyxoviruses hendra virus (hev) and nipah virus (niv) have been discovered [corrected]. objective: to examine, compare and contrast the functional characteristics of hmabs with the potential for prophylaxis and treatment of diseases caused by sars cov, hev and niv. methods: ...200919216624
case definitions for the 4 diseases requiring notification to who in all circumstances under the ihr (2005). 200919219965
interaction between sars-cov helicase and a multifunctional cellular protein (ddx5) revealed by yeast and mammalian cell two-hybrid systems.to reveal the putative cellular factors involved in sars coronavirus replication, the helicase (hel, nsp13) of sars coronavirus was used to screen the cdna library of rat pulmonary epithelial cells using the yeast two-hybrid system. positively interacting proteins were further tested using a mammalian cell hybrid system and co-immunoprecipitation in the human a549 cell line, which has been shown to support sars coronavirus replication. out of the seven positive clones observed by yeast two-hybri ...200919224332
aryl diketoacids (adk) selectively inhibit duplex dna-unwinding activity of sars coronavirus ntpase/helicase.as anti-hcv aryl diketoacids (adk) are good metal chelators, we anticipated that adks might serve as potential inhibitors of sars cov (scv) ntpase/helicase (hel) by mimicking the binding modes of the bismuth complexes which effectively competes for the zn(2+) ion binding sites in scv hel thereby disrupting and inhibiting both the ntpase and helicase activities. phosphate release assay and fret-based assay of the adk analogues showed that the adks selectively inhibit the duplex dna-unwinding acti ...200919233643
differential characteristics of the early stage of lung inflammation induced by sars-cov nucleocapsid protein related to age in the mouse.severe acute respiratory syndrome (sars) is an acute infectious disease of the respiratory system which has newly emerged. interestingly, it appears to be a disease that predominantly affects adults while the mortality in children is extremely low. however, the pathogenesis of sars in relation to different characteristics relevant to age remains unclear.200919234811
crossing barriers: infections of the lung and the gut.although known as respiratory pathogens, severe acute respiratory syndrome (sars) and its sister coronaviruses frequently cause enteric symptoms. in addition, other classically non-enteric viruses (such as hiv and influenza) may also have enteric effects that are crucial in their pathogeneses. these effects can be due to direct infection of the gut mucosa, but can also be because of decreased antibacterial defenses, increased mucosal permeability, bacterial translocation, and systemic leak of en ...200919129753
structural basis of inhibition specificities of 3c and 3c-like proteases by zinc-coordinating and peptidomimetic compounds.human coxsackievirus (cv) belongs to the picornavirus family, which consists of over 200 medically relevant viruses. in picornavirus, a chymotrypsin-like protease (3c(pro)) is required for viral replication by processing the polyproteins, and thus it is regarded as an antiviral drug target. a 3c-like protease (3cl(pro)) also exists in human coronaviruses (cov) such as 229e and the one causing severe acute respiratory syndrome (sars). to combat sars, we previously had developed peptidomimetic and ...200919144641
peptide nanoparticles as novel immunogens: design and analysis of a prototypic severe acute respiratory syndrome vaccine.severe acute respiratory syndrome (sars) is an infectious disease caused by a novel coronavirus that cost nearly 800 lives. while there have been no recent outbreaks of the disease, the threat remains as sars coronavirus (sars-cov) like strains still exist in animal reservoirs. therefore, the development of a vaccine against sars is in grave need. here, we have designed and produced a prototypic sars vaccine: a self-assembling polypeptide nanoparticle that repetitively displays a sars b-cell epi ...200919152635
severe acute respiratory syndrome coronavirus nsp9 dimerization is essential for efficient viral growth.the severe acute respiratory syndrome coronavirus (sars-cov) devotes a significant portion of its genome to producing nonstructural proteins required for viral replication. sars-cov nonstructural protein 9 (nsp9) was identified as an essential protein with rna/dna-binding activity, and yet its biological function within the replication complex remains unknown. nsp9 forms a dimer through the interaction of parallel alpha-helices containing the protein-protein interaction motif gxxxg. in order to ...200919153232
a novel replication-competent vaccinia vector mvtt is superior to mva for inducing high levels of neutralizing antibody via mucosal vaccination.mucosal vaccination offers great advantage for inducing protective immune response to prevent viral transmission and dissemination. here, we report our findings of a head-to-head comparison of two viral vectors modified vaccinia ankara (mva) and a novel replication-competent modified vaccinia tian tan (mvtt) for inducing neutralizing antibodies (nabs) via intramuscular and mucosal vaccinations in mice. mvtt is an attenuated variant of the wild-type vtt, which was historically used as a smallpox ...200919159014
the identification of a calmodulin-binding domain within the cytoplasmic tail of angiotensin-converting enzyme-2.angiotensin-converting enzyme (ace)-2 is a homolog of the well-characterized plasma membrane-bound angiotensin-converting enzyme. ace2 is thought to play a critical role in regulating heart function, and in 2003, ace2 was identified as a functional receptor for severe acute respiratory syndrome coronavirus. we have recently shown that like ace, ace2 undergoes ectodomain shedding and that this shedding event is up-regulated by phorbol esters. in the present study, we used gel shift assays to demo ...200919164471
the identification of a calmodulin-binding domain within the cytoplasmic tail of angiotensin-converting enzyme-2.angiotensin-converting enzyme (ace)-2 is a homolog of the well-characterized plasma membrane-bound angiotensin-converting enzyme. ace2 is thought to play a critical role in regulating heart function, and in 2003, ace2 was identified as a functional receptor for severe acute respiratory syndrome coronavirus. we have recently shown that like ace, ace2 undergoes ectodomain shedding and that this shedding event is up-regulated by phorbol esters. in the present study, we used gel shift assays to demo ...200919164471
delivery to the lower respiratory tract is required for effective immunization with newcastle disease virus-vectored vaccines intended for humans.newcastle disease virus (ndv), an avian virus, is being evaluated for the development of vectored human vaccines against emerging pathogens. previous studies of ndv-vectored vaccines in a mouse model suggested their potency after delivery by injection or by the intranasal route. we compared the efficacy of various routes of delivery of ndv-vectored vaccines in a non-human primate model. while delivery of an ndv-vectored vaccine by the combined intranasal/intratracheal route elicited protective i ...200919168110
crystal structures of the x-domains of a group-1 and a group-3 coronavirus reveal that adp-ribose-binding may not be a conserved property.the polyproteins of coronaviruses are cleaved by viral proteases into at least 15 nonstructural proteins (nsps). consisting of five domains, nsp3 is the largest of these (180-210 kda). among these domains, the so-called x-domain is believed to act as adp-ribose-1''-phosphate phosphatase or to bind poly(adp-ribose). however, here we show that the x-domain of infectious bronchitis virus (strain beaudette), a group-3 coronavirus, fails to bind adp-ribose. this is explained on the basis of the cryst ...200919177346
sars coronavirus spike protein-induced innate immune response occurs via activation of the nf-kappab pathway in human monocyte macrophages in vitro.a purified recombinant spike (s) protein was studied for its effect on stimulating human peripheral blood monocyte macrophages (pbmc). we examined inflammatory gene mrna abundances found in s protein-treated pbmc using gene arrays. we identified differential mrna abundances of genes with functional properties associated with antiviral (cxcl10) and inflammatory (il-6 and il-8) responses. we confirmed cytokine mrna increases by real-time quantitative(q) rt-pcr or elisa. we further analyzed the sen ...200919185596
comparative analysis of the immunogenicity of sars-cov nucleocapsid dna vaccine administrated with different routes in mouse model.the development of strategies to augment the immunogenicity of dna vaccines is critical for improving their clinical utility. one such strategy involves using the different immune routes with dna vaccines. in the present study, the immunogenicity of sars-cov nucleocapsid dna vaccine, induced by using the current routine vaccination routes (intramuscularly, by electroporation, or orally using live-attenuated salmonella typhimurium), was compared in mouse model. the comparison between the three va ...200919186202
the spike protein of sars-cov--a target for vaccine and therapeutic development.severe acute respiratory syndrome (sars) is a newly emerging infectious disease caused by a novel coronavirus, sars-coronavirus (sars-cov). the sars-cov spike (s) protein is composed of two subunits; the s1 subunit contains a receptor-binding domain that engages with the host cell receptor angiotensin-converting enzyme 2 and the s2 subunit mediates fusion between the viral and host cell membranes. the s protein plays key parts in the induction of neutralizing-antibody and t-cell responses, as we ...200919198616
globalisation and blood safety.globalisation may be viewed as the growing interdependence of countries worldwide through the increasing volume and variety of cross-border transactions in goods and services, and also through the more rapid and widespread diffusion of technology. globalisation is not just an economic phenomenon, although it is frequently described as such, but includes commerce, disease and travel, and immigration, and as such it affects blood safety and supply in various ways. the relatively short travel times ...200919081166
severe acute respiratory syndrome coronavirus protein 6 is required for optimal replication.severe acute respiratory syndrome coronavirus (sars-cov) encodes several accessory proteins of unknown function. one of these proteins, protein 6 (p6), which is encoded by orf6, enhances virus replication when introduced into a heterologous murine coronavirus (mouse hepatitis virus [mhv]) but is not essential for optimal sars-cov replication after infection at a relatively high multiplicity of infection (moi). here, we reconcile these apparently conflicting results by showing that p6 enhances sa ...200919091867
glycogen synthase kinase-3 regulates the phosphorylation of severe acute respiratory syndrome coronavirus nucleocapsid protein and viral replication.coronavirus (cov) nucleocapsid (n) protein is a highly phosphorylated protein required for viral replication, but whether its phosphorylation and the related kinases are involved in the viral life cycle is unknown. we found the severe acute respiratory syndrome cov n protein to be an appropriate system to address this issue. using high resolution page analysis, this protein could be separated into phosphorylated and unphosphorylated isoforms. mass spectrometric analysis and deletion mapping show ...200919106108
severe acute respiratory syndrome coronavirus triggers apoptosis via protein kinase r but is resistant to its antiviral activity.in this study, infection of 293/ace2 cells with severe acute respiratory syndrome coronavirus (sars-cov) activated several apoptosis-associated events, namely, cleavage of caspase-3, caspase-8, and poly(adp-ribose) polymerase 1 (parp), and chromatin condensation and the phosphorylation and hence inactivation of the eukaryotic translation initiation factor 2alpha (eif2alpha). in addition, two of the three cellular eif2alpha kinases known to be virus induced, protein kinase r (pkr) and pkr-like en ...200919109397
the pathogen receptor liver and lymph node sinusoidal endotelial cell c-type lectin is expressed in human kupffer cells and regulated by pu.1.human lsectin (liver and lymph node sinusoidal endothelial cell c-type lectin, clec4g) is a c-type lectin encoded within the l-sign/dc-sign/cd23 gene cluster. lsectin acts as a pathogen attachment factor for ebolavirus and the sars coronavirus, and its expression can be induced by interleukin-4 on monocytes and macrophages. although reported as a liver and lymph node sinusoidal endothelial cell-specific molecule, lsectin could be detected in the mutz-3 dendritic-like cell line at the messenger r ...200919111020
sars-cov proteins decrease levels and activity of human enac via activation of distinct pkc isoforms.among the multiple organ disorders caused by the severe acute respiratory syndrome coronavirus (sars-cov), acute lung failure following atypical pneumonia is the most serious and often fatal event. we hypothesized that two of the hydrophilic structural coronoviral proteins (s and e) would regulate alveolar fluid clearance by decreasing the cell surface expression and activity of amiloride-sensitive epithelial sodium (na(+)) channels (enac), the rate-limiting protein in transepithelial na(+) vect ...200919112100
severe acute respiratory syndrome-associated coronavirus infection in toronto children: a second look.during the severe acute respiratory syndrome outbreak of 2003, there was an impetus to provide clinical information to the medical community in a timely manner. accordingly, a preliminary report of our experience of suspected severe acute respiratory syndrome-associated coronavirus infections in children was published without microbiological findings. this report provides an update on pediatric severe acute respiratory syndrome-associated coronavirus infections in toronto, ontario, canada, that ...200919117866
[human coronaviruses].coronaviruses are a large group of viruses and infect a lot of species of mammals and birds. five coronaviruses currently infect humans: hcovs 229e and oc43, identified in the 1960s, sars-cov identified in march 2003 during the sars epidemic, and the hcovs nl63 and hku1, identified in 2004 and 2005 respectively. the genome of the coronaviruses is a linear, non-segmented, positive-sense single-stranded rna molecule of approximately 30kb. the evolution of these viruses occurs through some features ...200918456429
protease-mediated entry via the endosome of human coronavirus 229e.human coronavirus 229e, classified as a group i coronavirus, utilizes human aminopeptidase n (apn) as a receptor; however, its entry mechanism has not yet been fully elucidated. we found that hela cells infected with 229e via apn formed syncytia when treated with trypsin or other proteases but not in a low-ph environment, a finding consistent with syncytium formation by severe acute respiratory syndrome coronavirus (sars-cov). in addition, trypsin induced cleavage of the 229e s protein. by using ...200918971274
differential activities of cellular and viral macro domain proteins in binding of adp-ribose metabolites.macro domain is a highly conserved protein domain found in both eukaryotes and prokaryotes. macro domains are also encoded by a set of positive-strand rna viruses that replicate in the cytoplasm of animal cells, including coronaviruses and alphaviruses. the functions of the macro domain are poorly understood, but it has been suggested to be an adp-ribose-binding module. we have here characterized three novel human macro domain proteins that were found to reside either in the cytoplasm and nucleu ...200918983849
identification of a minimal peptide derived from heptad repeat (hr) 2 of spike protein of sars-cov and combination of hr1-derived peptides as fusion inhibitors.the heptad repeats (hr1 and hr2) of the spike protein of sars-cov are highly conserved regions forming a critical 6-helix bundle during the fusion step of virus entry and are attractive targets of entry inhibitors. in this study, we report that a minimal hr2 peptide, p6 of 23-mer, can block the fusion of sars-cov with an ic(50) of 1.04+/-0.22 microm. this finding supports the structural prediction of the deep groove of hr1 trimer as a target for fusion inhibitors, and suggests p6 as a potential ...200918983873
conserved amino acids w423 and n424 in receptor-binding domain of sars-cov are potential targets for therapeutic monoclonal antibody.the receptor-binding domain (rbd) on spike protein of severe acute respiratory syndrome-associated coronavirus (sars-cov) is the main region interacting with the viral receptor-ace2 and is a useful target for induction of neutralizing antibodies against sars-cov infection. here we generated two monoclonal antibodies (mabs), targeting rbd, with marked virus neutralizing activity. the mabs recognize a new conformational epitope which consists of several discontinuous peptides (aa. 343-367, 373-390 ...200918986662
crystal structures of two coronavirus adp-ribose-1''-monophosphatases and their complexes with adp-ribose: a systematic structural analysis of the viral adrp domain.the coronaviruses are a large family of plus-strand rna viruses that cause a wide variety of diseases both in humans and in other organisms. the coronaviruses are composed of three main lineages and have a complex organization of nonstructural proteins (nsp's). in the coronavirus, nsp3 resides a domain with the macroh2a-like fold and adp-ribose-1"-monophosphatase (adrp) activity, which is proposed to play a regulatory role in the replication process. however, the significance of this domain for ...200918987156
humoral and cellular immune responses induced by 3a dna vaccines against severe acute respiratory syndrome (sars) or sars-like coronavirus in mice.vaccine development for severe acute respiratory syndrome coronavirus (sars-cov) has mainly focused on the spike (s) protein. however, the variation of the s gene between viruses may affect the efficacy of a vaccine, particularly for cross-protection against sars-like cov (sl-cov). recently, a more conserved group-specific open reading frame (orf), the 3a gene, was found in both sars-cov and sl-cov. here, we studied the immunogenicity of human sars-cov 3a and bat sl-cov 3a dna vaccines in mice t ...200918987164
simple tests for rapid detection of canine parvovirus antigen and canine parvovirus-specific antibodies.canine parvovirus (cpv) is the number one viral cause of enteritis, morbidity, and mortality in 8-week-old young puppies. we have developed twin assays (slide agglutination test [sat] for cpv antigen and slide inhibition test [sit] for cpv antibody) that are sensitive, specific, cost-effective, generic for all genotypes of cpv, and provide instant results for cpv antigen detection in feces and antibody quantification in serum. we found these assays to be useful for routine applications in kennel ...200918987166
development of an enzyme-linked immunosorbent assay-based test with a cocktail of nucleocapsid and spike proteins for detection of severe acute respiratory syndrome-associated coronavirus-specific antibody.a new enzyme-linked immunosorbent assay (elisa)-based immunoglobulin g (igg)-plus-igm antibody detection test for severe acute respiratory syndrome (sars) has been developed by using a cocktail of four recombinant polypeptides as the antigen. these recombinant fragments were designed as parts of two different structural proteins from sars-associated coronavirus (sars-cov). one recombinant polypeptide, s251-683, was designed as part of the spike glycoprotein, and the other three polypeptides comp ...200919038782
severe acute respiratory syndrome (sars) coronavirus-induced lung epithelial cytokines exacerbate sars pathogenesis by modulating intrinsic functions of monocyte-derived macrophages and dendritic cells.severe acute respiratory syndrome (sars), which is caused by a novel coronavirus (cov), is a highly communicable disease with the lungs as the major pathological target. although sars likely stems from overexuberant host inflammatory responses, the exact mechanism leading to the detrimental outcome in patients remains unknown. pulmonary macrophages (mphi), airway epithelium, and dendritic cells (dc) are key cellular elements of the host innate defenses against respiratory infections. while pulmo ...200919004938
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