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biochemical and genetic analyses of murine hepatitis virus nsp15 endoribonuclease.the goal of this project was to better define the relationship between the endoribonuclease activity of murine hepatitis virus (mhv) nsp15 (mnsp15) and its role in virus infection. molecular modeling demonstrated that the catalytic residues of mnsp15 are superimposable with its severe acute respiratory syndrome coronavirus ortholog. alanine substitutions at three key residues in the mnsp15 catalytic pocket (h262, h277, and g275) and a double-mutant version (h262p and h277a) generated proteins wi ...200717898055
use of antibody avidity assays for diagnosis of severe acute respiratory syndrome coronavirus infection.an indirect immunofluorescent assay (euroimmun ag, luebeck, germany) was used to investigate the avidity of immunoglobulin g (igg), igm, iga, and total ig (iggam) antibody responses to severe acute respiratory syndrome coronavirus (sars cov) infections. serial serum samples from eight patients collected during the first, third, and ninth months after the onset of infection were evaluated. it was found that low-avidity igg antibodies were detected in 15/15 (100%), 1/5 (20%), and 0/8 (0%) serum sa ...200717881505
[expression of recombinant spike protein of sars-coronavirus in vaccinia virus and analysis of its immunogenicity].a recombinant vaccinia virus (rwr-sars-s)expressing spike protein of severe acute respiratory syndrome coronavirus was constructed. the expression of full length recombinant sars spike protein (rss) in hela cells possessing specific reaction ability to chicken anti-sera was confirmed by sds-page and western-blot (190 kd). hela cells infected with rwr-sars-s also showed high sensitivity in detecting specific serum antibody by indirect immunofluoresence assay (ifa). the results above indicated tha ...200717894231
[expression of recombinant spike protein of sars-coronavirus in vaccinia virus and analysis of its immunogenicity].a recombinant vaccinia virus (rwr-sars-s)expressing spike protein of severe acute respiratory syndrome coronavirus was constructed. the expression of full length recombinant sars spike protein (rss) in hela cells possessing specific reaction ability to chicken anti-sera was confirmed by sds-page and western-blot (190 kd). hela cells infected with rwr-sars-s also showed high sensitivity in detecting specific serum antibody by indirect immunofluoresence assay (ifa). the results above indicated tha ...200717894231
two-way antigenic cross-reactivity between severe acute respiratory syndrome coronavirus (sars-cov) and group 1 animal covs is mediated through an antigenic site in the n-terminal region of the sars-cov nucleoprotein.in 2002, severe acute respiratory syndrome-associated coronavirus (sars-cov) emerged in humans, causing a global epidemic. by phylogenetic analysis, sars-cov is distinct from known covs and most closely related to group 2 covs. however, no antigenic cross-reactivity between sars-cov and known covs was conclusively and consistently demonstrated except for group 1 animal covs. we analyzed this cross-reactivity by an enzyme-linked immunosorbent assay (elisa) and western blot analysis using specific ...200717913799
high prevalence of the cd14-159cc genotype in patients infected with severe acute respiratory syndrome-associated coronavirus.to investigate whether genetic factors of innate immunity might influence susceptibility and/or progression in individuals infected with sars, we evaluated the cd14 gene polymorphism in 198 hong kong blood donors and 152 hong kong severe acute respiratory syndrome (sars) patients who were previously genotyped for fcgammariia polymorphisms. the prevalence of the cd14-159cc polymorphism was significantly higher in the patients with severe sars than in the those with mild sars or controls (31% vers ...200717913858
severe acute respiratory syndrome coronavirus infection in vaccinated ferrets.development of vaccines to prevent severe acute respiratory syndrome (sars) is limited by the lack of well-characterized animal models. previous vaccine reports have noted robust neutralizing antibody and inflammatory responses in ferrets, resulting in enhanced hepatitis.200717922397
a novel subset of putative stem/progenitor cd34+oct-4+ cells is the major target for sars coronavirus in human lung.identification of the nature of severe acute respiratory syndrome (sars)-infected cells is crucial toward understanding the pathogenesis. using multicolor colocalization techniques, we previously reported that sars(+) cells in the lung of fatally infected patients expressed the only known functional receptor, angiotensin-converting enzyme 2, and also a binding receptor, liver/lymph node-specific icam-3-grabbing non-integrin (cd209l). in this study, we show that sars-infected cells also express t ...200717923501
the utility of preemptive mass influenza vaccination in controlling a sars outbreak during flu season.during flu season, respiratory infections can cause non-specific influenza-like-illnesses (ilis) in up to one-half of the general population. if a future sars outbreak were to coincide with flu season, it would become exceptionally difficult to distinguish sars rapidly and accurately from other ilis, given the non-specific clinical presentation of sars and the current lack of a widely available, rapid, diagnostic test. we construct a deterministic compartmental model to examine the potential imp ...200717924722
biochemical characterization of exoribonuclease encoded by sars coronavirus.the nsp14 protein is an exoribonuclease that is encoded by severe acute respiratory syndrome coronavirus (sars-cov). we have cloned and expressed the nsp14 protein in escherichia coli, and characterized the nature and the role(s) of the metal ions in the reaction chemistry. the purified recombinant nsp14 protein digested a 5'-labeled rna molecule, but failed to digest the rna substrate that is modified with fluorescein group at the 3'-hydroxyl group, suggesting a 3'-to-5' exoribonuclease activit ...200717927896
the 29-nucleotide deletion present in human but not in animal severe acute respiratory syndrome coronaviruses disrupts the functional expression of open reading frame 8.one of the most striking and dramatic genomic changes observed in the severe acute respiratory syndrome coronavirus (sars-cov) isolated from humans soon after its zoonotic transmission from palm civets was the acquisition of a characteristic 29-nucleotide deletion. this occurred in open reading frame 8 (orf8), one of the accessory genes unique to the sars-cov. the function of orf8 and the significance of the deletion are unknown. the intact orf8 present in animal and some early human isolates en ...200717928347
protection from infection with severe acute respiratory syndrome coronavirus in a chinese hamster model by equine neutralizing f(ab')2.to warrant potential clinical testing, the equine anti-severe acute respiratory syndrome coronavirus (sars-cov) f(ab')(2) requires evaluation in as many animal models as possible. in this study, we established a new animal model, the chinese hamster, susceptible to sars-cov infection. sars-cov could propagate effectively and sustain high levels for 1 wk in animal lungs. all animals were protected from sars-cov infection in preventive settings. further, when used therapeutically this antibody led ...200717931120
protection from infection with severe acute respiratory syndrome coronavirus in a chinese hamster model by equine neutralizing f(ab')2.to warrant potential clinical testing, the equine anti-severe acute respiratory syndrome coronavirus (sars-cov) f(ab')(2) requires evaluation in as many animal models as possible. in this study, we established a new animal model, the chinese hamster, susceptible to sars-cov infection. sars-cov could propagate effectively and sustain high levels for 1 wk in animal lungs. all animals were protected from sars-cov infection in preventive settings. further, when used therapeutically this antibody led ...200717931120
recent patents on treatment of severe acute respiratory syndrome (sars).severe acute respiratory syndrome (sars) is an epidemic that spread worldwide in early 2003. the aetiological agent was originally defined as a novel coronavirus and later designated as the sars coronavirus (sars-cov), which appears similar to other coronaviruses in both virion structure and genome organization with a single-stranded, plus-sense rna. however, the epidemiology and pathogenesis of sars remain poorly understood and there is currently no effective treatment. to date, considerable re ...200718221160
comparison of effectiveness of whole viral, n and n199 proteins by elisa for the rapid diagnosis of severe acute respiratory syndrome coronavirus.although severe acute respiratory syndrome (sars) has been controlled, the subsequently emerging sporadic cases in 2004 emphasize the necessity of developing a rapid diagnostic method, which would be of great help in clinical diagnosis and also wild host screening. this study aims to establish an effective and rapid serological tool for the diagnosis of sars-cov by comparison among whole viral, n and n199 proteins by elisa.200718167201
theoretically estimated risk of severe acute respiratory syndrome transmission through blood transfusion during an epidemic in shenzhen, guangdong, china in 2003.severe acute respiratory syndrome (sars) is a newly recognized infectious disease that caused an outbreak in south china in 2003. the cause of sars was identified as a novel coronavirus (cov). the existence of asymptomatic seroconvertors and the detection of the sars-cov rna in plasma during the course of infection all suggest that sars could, as least theoretically, be transmitted by transfusion. an estimate of the risk of sars transmission through blood transfusion will contribute to decisions ...200718036985
pathology and virus dispersion in cynomolgus monkeys experimentally infected with severe acute respiratory syndrome coronavirus via different inoculation routes.severe acute respiratory syndrome-associated coronavirus (sars-cov) causes sars. the pathogenic mechanisms of sars-cov remain poorly understood. six cynomolgus monkeys were inoculated with the hku39849 isolate of sars-cov via four routes. after intranasal inoculation, the virus was isolated from respiratory swabs on days 2-7 postinoculation (p.i.) and virus genome was detected in intestinal tissues on day 7 p.i. virus was not detected after intragastric inoculation. after intravenous inoculation ...200718039277
pathology and virus dispersion in cynomolgus monkeys experimentally infected with severe acute respiratory syndrome coronavirus via different inoculation routes.severe acute respiratory syndrome-associated coronavirus (sars-cov) causes sars. the pathogenic mechanisms of sars-cov remain poorly understood. six cynomolgus monkeys were inoculated with the hku39849 isolate of sars-cov via four routes. after intranasal inoculation, the virus was isolated from respiratory swabs on days 2-7 postinoculation (p.i.) and virus genome was detected in intestinal tissues on day 7 p.i. virus was not detected after intragastric inoculation. after intravenous inoculation ...200718039277
discontinued drugs in 2006: anti-infectives.of the drugs dropped from development in 2006, 11 were being developed for infectious diseases. of these, nine were for viral diseases, including four against hiv, two against hepatitis c virus and one each against respiratory syncytial virus, severe acute respiratory syndrome (coronavirus) and a variety of viruses. the nine antiviral agents comprised six synthetic small-molecule compounds, one peptide, one monoclonal antibody and a vaccine. the remaining two agents were a vaccine for pseudomona ...200718041997
[optimization of expression condition of sars-cov pups genes in e. coli].according to previous studies of sars-cov (severe acute respiratory syndrome coronavirus), a variety of novel accessory genes have been identified in sars-cov genome, which were interspersed the structural genes of sars-cov and considered to be unique to the sars-cov genome. the predicted unknown proteins (pups) encoded by the accessory genes might play important roles in the sars-cov infection. three of those genes, called x4, x5 and orf10, were synthesized and introduced into e. coli to induce ...200718050746
severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection.before the emergence of severe acute respiratory syndrome (sars) coronavirus (sars-cov) in 2003, only 12 other animal or human coronaviruses were known. the discovery of this virus was soon followed by the discovery of the civet and bat sars-cov and the human coronaviruses nl63 and hku1. surveillance of coronaviruses in many animal species has increased the number on the list of coronaviruses to at least 36. the explosive nature of the first sars epidemic, the high mortality, its transient reeme ...200717934078
co-circulation of human metapneumovirus and sars-associated coronavirus during a major nosocomial sars outbreak in hong kong.the clinico-epidemiological significance of human metapneumovirus (hmpv) detected during the sars outbreak is unknown.200717936066
human monoclonal antibodies by immortalization of memory b cells.the administration of hyper immune sera to prevent or treat life-threatening infections is a remarkable milestone in medicine and biotechnology that has been achieved more than a century ago. yet, the therapeutic use of monoclonal antibodies in this field has developed slowly over the last decades. here we compare and contrast current methods to generate human monoclonal antibodies and highlight the advantages of exploiting the human antibody repertoire using a novel method that allows efficient ...200718063358
heterologous expression of sars-cov orf10 and x5 genes in e. coli and streptomyces lividans tk24.in previous studies a variety of novel accessory genes has been identified that were interspersed among the structural genes of the sars-cov (severe acute respiratory syndrome coronavirus) genome. the predicted unknown proteins (pups) encoded by the accessory genes, which are considered to be unique to the sars-cov genome, might play important roles in the sars-cov infection. two of these genes, called orf10 and x5, were synthesized and introduced into e. coli and streptomyces lividans tk24, res ...200718069252
[development and application of a safe sars-cov neutralization assay based on lentiviral vectors pseudotyped with sars-cov spike protein].the severe acute respiratory syndrome-associated coronavirus (sars-cov) spike protein (s) is a major target for neutralizing antibody. to develop and apply a safe neutralization assay for sars-cov, lentiviral sars-cov s pseudotypes had been constructed based on a three plasmid system, which contained pvrc8304 (harboring codon optimized full-length sars-cov s protein), pcmv delta 8. 2 (hiv-1 gag/pol construct) and phr'cmv egfp (the green fluorescent protein reporter construct). the pseudo-typed l ...200718092680
duration of antibody responses after severe acute respiratory syndrome.among 176 patients who had had severe acute respiratory syndrome (sars), sars-specific antibodies were maintained for an average of 2 years, and significant reduction of immunoglobulin g-positive percentage and titers occurred in the third year. thus, sars patients might be susceptible to reinfection >or=3 years after initial exposure.200718258008
genomic analysis and geographic visualization of h5n1 and sars-cov.emerging infectious diseases and organisms present critical issues of national security public health, and economic welfare. we still understand little about the zoonotic potential of many viruses. to this end, we are developing novel database tools to manage comparative genomic datasets. these tools add value because they allow us to summarize the direction, frequency and order of genomic changes. we will perform numerous real world tests with our tools with both avian influenza and coronavirus ...200718694075
intratracheal inoculation of severe acute respiratory syndrome coronavirus in monkeys macaca rhesus.an animal model for infection with severe acute respiratory syndrome coronavirus (sarscov) was evaluated in monkeys macaca rhesus. the monkeys were inoculated into the trachea with ns-i strain of sars-cov and the clinical manifestation of the illness was monitored. the clinical samples collected from infected monkeys were examined by immumnofluorescence assay (ifa), pathological inspection, rtpcr, and by virus isolation. the infected animals demonstrated mild clinical symptoms including fever. t ...200718076307
structural basis of mercury- and zinc-conjugated complexes as sars-cov 3c-like protease inhibitors.five active metal-conjugated inhibitors (pma, tdt, epdtc, jmf1586 and jmf1600) bound with the 3c-like protease of severe acute respiratory syndrome (sars)-associated coronavirus were analyzed crystallographically. the complex structures reveal two major inhibition modes: hg(2+)-pma is coordinated to c(44), m(49) and y(54) with a square planar geometry at the s3 pocket, whereas each zn(2+) of the four zinc-inhibitors is tetrahedrally coordinated to the h(41)-c(145) catalytic dyad. for anti-sars d ...200717981158
application of proteinchip array profiling in serum biomarker discovery for patients suffering from severe acute respiratory syndrome.a new strain of coronavirus has caused an outbreak of severe acute respiratory syndrome (sars) from 2002 to 2003 resulting in 774 deaths worldwide. by protein chip array profiling technology, a number of serum biomarkers that might be useful in monitoring the clinical course of sars patients were identified. this book chapter describes how the protein chip array profiling was carried out for this study. briefly, sars patients' serum samples were first fractionated in q ceramic hyperd ion exchang ...200718220240
progress in anti-sars coronavirus chemistry, biology and chemotherapy. 200719649165
nmr assignment of the domain 513-651 from the sars-cov nonstructural protein nsp3.sequence-specific nmr assignments of an internal domain of the protein nsp3, nsp3(513-651), which is a part of the sars coronavirus (sars-cov) replicase polyprotein, have been determined, using triple-resonance nmr experiments with the uniformly [(13)c,(15)n]-labeled protein. the complete assignments (>99%) provide the basis for the ongoing three-dimensional structure determination.200719636862
identification of phosphorylation sites in the nucleocapsid protein (n protein) of sars-coronavirus.after decoding the genome of sars-coronavirus (sars-cov), next challenge is to understand how this virus causes the illness at molecular bases. of the viral structural proteins, the n protein plays a pivot role in assembly process of viral particles as well as viral replication and transcription. the sars-cov n proteins expressed in the eukaryotes, such as yeast and hek293 cells, appeared in the multiple spots on two-dimensional electrophoresis (2de), whereas the proteins expressed in e. coli sh ...200732288628
bismuth complexes inhibit the sars coronavirus.festgehalten: bismutkomplexe, darunter ranitidinbismutcitrat, inhibieren die nucleosidtriphosphat‐hydrolase‐ und dna‐entfaltungsaktivitäten der sars‐coronavirus(scv)‐helicase effektiv und reduzieren das ausmaß der scv‐replikation in infizierten zellen erheblich. es lohnt sich also, wirkstoffe auf bismutbasis für die behandlung von scv‐infektionen in vivo weiter zu untersuchen. ss=einzelstrang‐dna, ds=doppelstrang‐dna.200732313314
fatalness of virus depends upon its cell fractal geometry.why do more complex viruses (e.g., hiv, aids-virus and sars coronavirus) tend to be more fatal? the paper concludes that the cell fractal geometry of viruses is the key. this paper also suggests two possible new approaches using nanotechnology and temperature to cure or prevent virus infection.200832288359
stigmatized ethnicity, public health, and globalization.the prejudicial linking of infection with ethnic minority status has a long-established history, but in some ways this association may have intensified under the contemporary circumstances of the "new public health" and globalization. this study analyzes this conflation of ethnicity and disease victimization by considering the stigmatization process that occurred during the 2003 outbreak of severe acute respiratory syndrome (sars) in toronto. the attribution of stigma during the sars outbreak oc ...200821847845
nmr assignment of the nonstructural protein nsp3(1066-1181) from sars-cov.sequence-specific nmr assignments of the globular core comprising the residues 1066-1181 within the non-structural protein nsp3e from the sars coronavirus have been obtained using triple-resonance nmr experiments with the uniformly [(13)c, (15)n]-labeled protein. the backbone and side chain assignments are nearly complete, providing the basis for the ongoing nmr structure determination. a preliminary identification of regular secondary structures has been derived from the (13)c chemical shifts.200819636888
broadening of neutralization activity to directly block a dominant antibody-driven sars-coronavirus evolution pathway.phylogenetic analyses have provided strong evidence that amino acid changes in spike (s) protein of animal and human sars coronaviruses (sars-covs) during and between two zoonotic transfers (2002/03 and 2003/04) are the result of positive selection. while several studies support that some amino acid changes between animal and human viruses are the result of inter-species adaptation, the role of neutralizing antibodies (nabs) in driving sars-cov evolution, particularly during intra-species transm ...200818989460
structural and biochemical investigation of heptad repeat derived peptides of human sars corona virus (hsars-cov) spike protein.hsars-cov is the causative agent for sars infection. its spike glycoprotein (s) is processed by host furin enzyme to produce s1 and s2 fragments, the latter being crucial for fusion with the host membrane. this takes place via formation of a coiled coil 6-helix bundle involving n and c-terminal heptad repeat domains (hr-n and hr-c) of s2. several fluorescent and non-fluorescent peptides from these domains were synthesized to examine their interactions by circular dichroism, thermal denaturation, ...200818991761
difference in receptor usage between severe acute respiratory syndrome (sars) coronavirus and sars-like coronavirus of bat origin.severe acute respiratory syndrome (sars) is caused by the sars-associated coronavirus (sars-cov), which uses angiotensin-converting enzyme 2 (ace2) as its receptor for cell entry. a group of sars-like covs (sl-covs) has been identified in horseshoe bats. sl-covs and sars-covs share identical genome organizations and high sequence identities, with the main exception of the n terminus of the spike protein (s), known to be responsible for receptor binding in covs. in this study, we investigated the ...200818077725
isolation of inhibitory rna aptamers against severe acute respiratory syndrome (sars) coronavirus ntpase/helicase.recent outbreak of severe acute respiratory syndrome (sars) that caused almost 800 victims requires a development of efficient inhibitor against sars coronavirus (scv). in this study, rna aptamers against scv ntpase/helicase (nsp10) were isolated from rna library containing random sequences of 40 nts using in vitro selection technique. nucleotide sequences of enriched rna aptamer pool (es15 rna) contain ag-rich conserved sequence of 10-11 nucleotides [aaaggr(g)gaag; r, purine base] and/or additi ...200818082623
turkey coronavirus non-structure protein nsp15--an endoribonuclease.turkey coronavirus (tcov) polyprotein was predicted to be cleaved into 15 non-structural proteins (nsp2 to nsp16), but none of these nsps have been characterized. tcov nsp15 consists of 338 residues and shares 40% sequence similarity to u-specific nidovirales endoribonuclease (nendou) of severe acute respiratory syndrome coronavirus. objective: the purpose of the present study was to characterize tcov nsp15. methods: the tcov nsp15 gene was cloned into ptriex1 and expressed as a c-terminal his-t ...200819023218
differential inhibitory activities and stabilisation of dna aptamers against the sars coronavirus helicase.the helicase from severe acute respiratory syndrome coronavirus (sars-cov) possesses ntpase, duplex rna/dna-unwinding and rna-capping activities that are essential for viral replication and proliferation. here, we have isolated dna aptamers against the sars-cov helicase from a combinatorial dna library. these aptamers show two distinct classes of secondary structure, g-quadruplex and non-g-quadruplex, as shown by circular dichroism and gel electrophoresis. all of the aptamers that were selected ...200819031435
synthetic recombinant bat sars-like coronavirus is infectious in cultured cells and in mice.defining prospective pathways by which zoonoses evolve and emerge as human pathogens is critical for anticipating and controlling both natural and deliberate pandemics. however, predicting tenable pathways of animal-to-human movement has been hindered by challenges in identifying reservoir species, cultivating zoonotic organisms in culture, and isolating full-length genomes for cloning and genetic studies. the ability to design and recover pathogens reconstituted from synthesized cdnas has the p ...200819036930
the expression and antigenicity of a truncated spike-nucleocapsid fusion protein of severe acute respiratory syndrome-associated coronavirus.in the absence of effective drugs, controlling sars relies on the rapid identification of cases and appropriate management of the close contacts, or effective vaccines for sars. therefore, developing specific and sensitive laboratory tests for sars as well as effective vaccines are necessary for national authorities.200819038059
seroprevalence of sars coronavirus among residents near a hospital with a nosocomial outbreak.an epidemic of severe acute respiratory syndrome (sars) occurred in taiwan from april to july 2003. a nosocomial outbreak of sars occurred at kaohsiung chang gung memorial hospital (cgmh) in may 2003. the purpose of our study was to survey the prevalence of the sars coronavirus (cov) in a community adjacent to kaohsiung cgmh and collect demographic data, including basic information about health status, household, and possible risk factors for sars-cov infection.200818971158
design, synthesis and screening of antisense peptide based combinatorial peptide libraries towards an aromatic region of sars-cov.a combination of high-performance affinity chromatography and antisense peptide based combinatorial peptide libraries was used to screen a potential inhibitor for sars-cov. an aromatic-amino acid-rich region within the transmembrane domain at the c terminal of spike (s) protein identified as a membrane-active region was chosen as the target sense peptide (sp) and immobilized as affinity ligand. four antisense peptides were designed based on the degeneracy of genetic codes. one of them was screen ...200818383098
sars vaccine based on a replication-defective recombinant vesicular stomatitis virus is more potent than one based on a replication-competent vector.a sars vaccine based on a live-attenuated vesicular stomatitis virus (vsv) recombinant expressing the sars-cov s protein provides long-term protection of immunized mice from sars-cov infection (kapadia, s.u., rose, j. k., lamirande, e., vogel, l., subbarao, k., roberts, a., 2005. long-term protection from sars coronavirus infection conferred by a single immunization with an attenuated vsv-based vaccine. virology 340(2), 174-82.). because it is difficult to obtain regulatory approval of vaccine b ...200818396306
interferon alfacon 1 inhibits sars-cov infection in human bronchial epithelial calu-3 cells.the primary targets for sars-cov infection are the epithelial cells in the respiratory and intestinal tract. the angiotensin-converting enzyme 2 (ace-2) has been identified as a functional receptor for sars-cov. ace-2 has been shown to be expressed at the apical domain of polarized calu-3 cells. in this report, interferon alfacon 1 was examined for inhibitory activities against sars-cov on human lung carcinoma epithelial calu-3 cell line and the other three african green monkey kidney epithelial ...200818406349
heteroaromatic ester inhibitors of hepatitis a virus 3c proteinase: evaluation of mode of action.the related 3c and 3c-like proteinase (3c(pro) and 3cl(pro)) of picornaviruses and coronaviruses, respectively, are good drug targets. as part of an effort to generate broad-spectrum inhibitors of these enzymes, we screened a library of inhibitors based on a halopyridinyl ester from a previous study of the severe acute respiratory syndrome (sars) 3cl proteinase against hepatitis a virus (hav) 3c(pro). three of the compounds, which also had furan rings, inhibited the cleavage activity of hav 3c(p ...200818407505
coronavirus nonstructural protein 16 is a cap-0 binding enzyme possessing (nucleoside-2'o)-methyltransferase activity.the coronavirus family of positive-strand rna viruses includes important pathogens of livestock, companion animals, and humans, including the severe acute respiratory syndrome coronavirus that was responsible for a worldwide outbreak in 2003. the unusually complex coronavirus replicase/transcriptase is comprised of 15 or 16 virus-specific subunits that are autoproteolytically derived from two large polyproteins. in line with bioinformatics predictions, we now show that feline coronavirus (fcov) ...200818417574
genomic characterizations of bat coronaviruses (1a, 1b and hku8) and evidence for co-infections in miniopterus bats.we previously reported the detection of bat coronaviruses (bat covs 1a, 1b, hku7, hku8 and bat-severe acute respiratory syndrome coronavirus) in miniopterus spp. that cohabit a cave in hong kong. here, we report the full genomic sequences of bat covs 1a, 1b and hku8. bat covs 1a and 1b, which are commonly found in the miniopterus, are phylogenetically closely related. using species-specific rt-pcr assays, bat covs 1a and 1b were confirmed to have distinct host specificities to miniopterus magnat ...200818420807
genomic characterizations of bat coronaviruses (1a, 1b and hku8) and evidence for co-infections in miniopterus bats.we previously reported the detection of bat coronaviruses (bat covs 1a, 1b, hku7, hku8 and bat-severe acute respiratory syndrome coronavirus) in miniopterus spp. that cohabit a cave in hong kong. here, we report the full genomic sequences of bat covs 1a, 1b and hku8. bat covs 1a and 1b, which are commonly found in the miniopterus, are phylogenetically closely related. using species-specific rt-pcr assays, bat covs 1a and 1b were confirmed to have distinct host specificities to miniopterus magnat ...200818420807
a triffic perspective on acute lung injury.acute lung injury (ali) is a leading cause of death in people infected with h5n1 avian influenza virus or the sars-coronavirus. imai et al. (2008) now report that ali is triggered by the signaling of oxidized phospholipids through toll-like receptor 4 (tlr4) and the adaptor protein trif. these findings provide insight into the molecular pathogenesis of ali, a condition for which treatment options are currently very limited.200818423191
is the anti-psychotic, 10-(3-(dimethylamino)propyl)phenothiazine (promazine), a potential drug with which to treat sars infections? lack of efficacy of promazine on sars-cov replication in a mouse model.phenothiazine and derivatives were tested for inhibition of sars-cov replication. phenothiazine slightly inhibited sars-cov replication in a neutral red (nr) uptake assay. adding a propylamino group to give promazine reduced virus yields (vyr assay) with an ec(90)=8.3+/-2.8 microm, but without selectivity. various substitutions in the basic phenothiazine structure did not promote efficacy. phenazine ethosulfate was the most potent compound by vyr assay (ec(90)=6.1+/-4.3 microm). all compounds we ...200818423639
importance of sars-cov spike protein trp-rich region in viral infectivity.sars-cov entry is mediated by spike glycoprotein. during the viral and host cellular membrane fusion, hr1 and hr2 form 6-helix bundle, positioning the fusion peptide closely to the c-terminal region of ectodomain to drive apposition and subsequent membrane fusion. connecting to the hr2 region is a trp-rich region which is absolutely conserved in members of coronaviruses. to investigate the importance of trp-rich region in sars-cov entry, we produced different mutated s proteins using alanine sca ...200818424264
clinical features, pathogenesis and immunobiology of severe acute respiratory syndrome.severe acute respiratory syndrome coronavirus is a novel virus responsible for the major pandemic in 2003, and it re-emerged in china in late 2003 and 2004 following resumption of wild animal trading activities. over the past few years, research work has looked into factors that may lead to super-spreading events, clinical/laboratory parameters that may differentiate severe acute respiratory syndrome from other causes of community-acquired pneumonia, the origin of severe acute respiratory syndro ...200818427248
identification of a novel transcriptional repressor (hepis) that interacts with nsp-10 of sars coronavirus.a novel gene was previously isolated from a cdna library of human embryo lung tissue by its encoded protein, which interacts with non-structural protein 10 (nsp-10) of the severe acute respiratory syndrome coronavirus (sars-cov). the protein was named human embryo lung cellular protein interacting with sars-cov nsp-10 (hepis), and it is composed of 147 amino acids with several ck ii phosphorylation sites. in the present study, we demonstrated that hepis was capable of suppressing chloramphenicol ...200818433331
search for potential target site of nucleocapsid gene for the design of an epitope-based sars dna vaccine.it is believed today that nucleocapsid protein (n) of severe acute respiratory syndrome (sars)-cov is one of the most promising antigen candidates for vaccine design. in this study, three fragments [n1 (residues: 1-422); n2 (residues: 1-109); n3 (residues: 110-422)] of n protein of sars-cov were expressed in escherichia coli and analyzed by pooled sera of convalescence phase of sars patients. three gene fragments [n1 (1-1269 nt), n2 (1-327 nt) and n3 (328-1269 nt)-expressing the same proteins of ...200818440652
fusion core structure of the severe acute respiratory syndrome coronavirus (sars-cov): in search of potent sars-cov entry inhibitors.severe acute respiratory coronavirus (sars-cov) spike (s) glycoprotein fusion core consists of a six-helix bundle with the three c-terminal heptad repeat (hr2) helices packed against a central coiled-coil of the other three n-terminal heptad repeat (hr1) helices. each of the three peripheral hr2 helices shows prominent contacts with the hydrophobic surface of the central hr1 coiled-coil. the concerted protein-protein interactions among the hr helices are responsible for the fusion event that lea ...200818442051
evaluation of cotton rats as a model for severe acute respiratory syndrome.experimental studies were conducted to evaluate two species of cotton rats, sigmodon hispidus and sigmodon fulviventer, as a model for severe acute respiratory syndrome (sars). blood and turbinate wash samples, and lung tissue were collected from each animal at different time points after sars coronavirus (cov) infection for determining the growth curve of virus, if any, by the standard infectivity assay in vero e6 cells. in addition, sections of the lung, liver, spleen, and kidney were taken an ...200818447621
evaluation of cotton rats as a model for severe acute respiratory syndrome.experimental studies were conducted to evaluate two species of cotton rats, sigmodon hispidus and sigmodon fulviventer, as a model for severe acute respiratory syndrome (sars). blood and turbinate wash samples, and lung tissue were collected from each animal at different time points after sars coronavirus (cov) infection for determining the growth curve of virus, if any, by the standard infectivity assay in vero e6 cells. in addition, sections of the lung, liver, spleen, and kidney were taken an ...200818447621
what caused lymphopenia in sars and how reliable is the lymphokine status in glucocorticoid-treated patients?severe acute respiratory syndrome (sars) outbreak in 2002-03 caused morbidity in over 8000 individuals and mortality in 744 in 29 countries. lymphopenia along with neutrophilia was a feature of sars, as it is in respiratory syncytial virus (rsv) and ebola infections, to name a few. direct infestation of lymphocytes, neutrophils and macrophages by sars coronavirus (cov) has been debated as a cause of lymphopenia, but there is no convincing data. lymphopenia can be caused by glucocorticoids, and t ...200818448259
the nucleocapsid protein of severe acute respiratory syndrome coronavirus inhibits cell cytokinesis and proliferation by interacting with translation elongation factor 1alpha.severe acute respiratory syndrome coronavirus (sars-cov) is the etiological agent of sars, an emerging disease characterized by atypical pneumonia. using a yeast two-hybrid screen with the nucleocapsid (n) protein of sars-cov as a bait, the c terminus (amino acids 251 to 422) of the n protein was found to interact with human elongation factor 1-alpha (ef1alpha), an essential component of the translational machinery with an important role in cytokinesis, promoting the bundling of filamentous acti ...200818448518
severe acute respiratory syndrome coronavirus protein 6 accelerates murine hepatitis virus infections by more than one mechanism.the severe acute respiratory syndrome coronavirus (sars-cov) encodes numerous accessory proteins whose importance in the natural infection process is currently unclear. one of these accessory proteins is set apart by its function in the context of a related murine hepatitis virus (mhv) infection. sars-cov protein 6 increases mhv neurovirulence and accelerates mhv infection kinetics in tissue culture. protein 6 also blocks nuclear import of macromolecules from the cytoplasm, a process known to in ...200818448520
structural analysis of major species barriers between humans and palm civets for severe acute respiratory syndrome coronavirus infections.it is believed that a novel coronavirus, severe acute respiratory syndrome coronavirus (sars-cov), was passed from palm civets to humans and caused the epidemic of sars in 2002 to 2003. the major species barriers between humans and civets for sars-cov infections are the specific interactions between a defined receptor-binding domain (rbd) on a viral spike protein and its host receptor, angiotensin-converting enzyme 2 (ace2). in this study a chimeric ace2 bearing the critical n-terminal helix fro ...200818448527
the discovery of angiotensin-converting enzyme 2 and its role in acute lung injury in mice.during several months of 2002, severe acute respiratory syndrome (sars) caused by sars-coronavirus (sars-cov) spread rapidly from china throughout the world, causing more than 800 deaths due to the development of acute respiratory distress syndrome (ards), which is the severe form of acute lung injury (ali). interestingly, a novel homologue of angiotensin-converting enzyme, termed angiotensin-converting enzyme 2 (ace2), has been identified as a receptor for sars-cov. angiotensin-converting enzym ...200818448662
sirna silencing of angiotensin-converting enzyme 2 reduced severe acute respiratory syndrome-associated coronavirus replications in vero e6 cells.the outbreak of severe acute respiratory syndrome (sars) in 2002-2003 has had a significant impact worldwide. no effective prophylaxis or treatment for sars is available up to now. angiotensin-converting enzyme 2 (ace2) is the cellular receptor for sars-associated coronavirus (sars-cov). by expressing a u6 promoter-driven small interfering rna containing sequences homologous to part of ace2 mrna, we successfully silenced ace2 expression in vero e6 cells. by detecting negative strand sars-cov rna ...200818449585
sars-coronavirus replication/transcription complexes are membrane-protected and need a host factor for activity in vitro.sars-coronavirus (sars-cov) replication and transcription are mediated by a replication/transcription complex (rtc) of which virus-encoded, non-structural proteins (nsps) are the primary constituents. the 16 sars-cov nsps are produced by autoprocessing of two large precursor polyproteins. the rtc is believed to be associated with characteristic virus-induced double-membrane structures in the cytoplasm of sars-cov-infected cells. to investigate the link between these structures and viral rna synt ...200818451981
pathogenicity of severe acute respiratory coronavirus deletion mutants in hace-2 transgenic mice.recombinant severe acute respiratory virus (sars-cov) variants lacking the group specific genes 6, 7a, 7b, 8a, 8b and 9b (rsars-cov-delta[6-9b]), the structural gene e (rsars-cov-deltae), and a combination of both sets of genes (rsars-cov-delta[e,6-9b]) have been generated. all these viruses were rescued in monkey (vero e6) cells and were also infectious for human (huh-7, huh7.5.1 and caco-2) cell lines and for transgenic (tg) mice expressing the sars-cov receptor human angiotensin converting en ...200818452964
mechanisms of severe acute respiratory syndrome pathogenesis and innate immunomodulation.the modulation of the immune response is a common practice of many highly pathogenic viruses. the emergence of the highly pathogenic coronavirus severe acute respiratory virus (sars-cov) serves as a robust model system to elucidate the virus-host interactions that mediate severe end-stage lung disease in humans and animals. coronaviruses encode the largest positive-sense rna genome of approximately 30 kb, encode a variety of replicase and accessory open reading frames that are structurally uniqu ...200819052324
characteristic features and outcomes of severe acute respiratory syndrome found in severe acute respiratory syndrome intensive care unit patients.the aim of the study was to identify characteristic clinical features and outcomes of critically ill patients with confirmed severe acute respiratory syndrome (sars).200819056023
detection of sars coronavirus in humans and animals by conventional and quantitative (real time) reverse transcription polymerase chain reactions.severe acute respiratory syndrome is a novel human disease caused by a coronavirus of animal origin. soon after the discovery sars-cov, several molecular assays were described for the detection of this virus. of these, conventional and quantitative rt-pcr approaches were the primary tools for sars-cov rna detection. in this chapter we describe a two-step conventional rt-pcr and a one-step quantitative rt-pcr that were used routinely in our laboratories during the sars outbreak.200819057863
pseudotyped vesicular stomatitis virus for analysis of virus entry mediated by sars coronavirus spike proteins.severe acute respiratory syndrome (sars) coronavirus (cov) contains a spike (s) protein that binds to a receptor molecule (angiotensin-converting enzyme 2; ace2), induces membrane fusion, and serves as a neutralizing epitope. to study the functions of the s protein, we describe here the generation of sars-cov s protein-bearing vesicular stomatitis virus (vsv) pseudotype using a vsvdeltag*/gfp system in which the g gene is replaced by the green fluorescent protein (gfp) gene (vsv-sars-cov-st19/gf ...200819057867
engineering infectious cdnas of coronavirus as bacterial artificial chromosomes.the construction of coronavirus (cov) infectious clones had been hampered by the large size of the viral genome (around 30kb) and the instability of plasmids carrying cov replicase sequences in escherichia coli. several approaches have been developed to overcome these problems. here we describe the engineering of cov full-length cdna clones using bacterial artificial chromosomes (bacs). in this system the viral rna is expressed in the cell nucleus under the control of the cytomegalovirus promote ...200819057870
detection of group 1 coronaviruses in bats using universal coronavirus reverse transcription polymerase chain reactions.the zoonotic transmission of sars coronavirus from animals to humans revealed the potential impact of coronaviruses on mankind. this incident also triggered several surveillance programs to hunt for novel coronaviruses in human and wildlife populations. using classical rt-pcr assays that target a highly conserved sequence among coronaviruses, we identified the first coronaviruses in bats. these assays and the cloning and sequencing of the pcr products are described in this chapter. using the sam ...200819057871
generation of recombinant coronaviruses using vaccinia virus as the cloning vector and stable cell lines containing coronaviral replicon rnas.coronavirus reverse genetic systems have become valuable tools for studying the molecular biology of coronavirus infections. they have been applied to the generation of recombinant coronaviruses, selectable replicon rnas, and coronavirus-based vectors for heterologous gene expression. here we provide a collection of protocols for the generation, cloning, and modification of full-length coronavirus cdna using vaccinia virus as a cloning vector. based on cloned coronaviral cdna, we describe the ge ...200819057873
establishment and characterization of monoclonal antibodies against sars coronavirus.immunological detection of viruses and their components by monoclonal antibodies is a powerful method for studying the structure and function of viral molecules. here we describe detailed methods for establishing monoclonal antibodies against severe acute respiratory syndrome coronavirus (sars-cov). b cell hybridomas are generated from mice that are hyperimmunized with inactivated sars-cov virions. the hybridomas produce monoclonal antibodies that recognize viral component molecules, including t ...200819057876
large-scale preparation of uv-inactivated sars coronavirus virions for vaccine antigen.in general, a whole virion serves as a simple vaccine antigen and often essential material for the analysis of immune responses against virus infection. however, to work with highly contagious pathogens, it is necessary to take precautions against laboratory-acquired infection. we have learned many lessons from the recent outbreak of severe acute respiratory syndrome (sars). in order to develop an effective vaccine and diagnostic tools, we prepared uv-inactivated sars coronavirus on a large scal ...200819057880
school model and new targeting strategies.protein-protein interactions play a central role in biological processes and thus are an appealing target for innovative drug design a nd development. they can be targeted bysmall molecule inhibitors, peptides and peptidomimetics, which represent an alternative to protein therapeutics that carry many disadvantages. in this chapter, i describe specific protein-protein interactions suggested by a novel model of immune signaling, the signaling chain homooligomerization (school) model, to be critica ...200819065798
sars- and other coronaviruses. 200819068525
myd88 is required for protection from lethal infection with a mouse-adapted sars-cov.a novel human coronavirus, sars-cov, emerged suddenly in 2003, causing approximately 8000 human cases and more than 700 deaths worldwide. since most animal models fail to faithfully recapitulate the clinical course of sars-cov in humans, the virus and host factors that mediate disease pathogenesis remain unclear. recently, our laboratory and others developed a recombinant mouse-adapted sars-cov (rma15) that was lethal in balb/c mice. in contrast, intranasal infection of young 10-week-old c57bl/6 ...200819079579
analysis of severe acute respiratory syndrome coronavirus structural proteins in virus-like particle assembly.sars-cov has four major structural proteins: the n, s, m, and e proteins. to investigate the mechanism of sars-cov assembly, we cloned the genes encoding these four proteins into the eukaryotic expression vector pcaggs and transfected them into 293t cells. when all four expression vectors were co-transfected vlp formed, as confirmed using electron microscopy. using a rabbit polyclonal antibody specific to the n protein, n-protein-containing particles similar in size to the vlp were also observed ...200819120977
molecular targets for diagnostics and therapeutics of severe acute respiratory syndrome (sars-cov).the large number of deaths in a short period of time due to the spread of severe acute respiratory syndrome (sars) infection led to the unparalleled collaborative efforts world wide to determine and characterize the new coronavirus (sars-cov). the full genome sequence was determined within weeks of the first outbreak by the canadian group with international collaboration. as per the world health organization (who), the continual lack of a rapid laboratory test to aid the early diagnosis of suspe ...200819203466
[expression, purification and antibody preparation of recombinat sars-cov x5 protein].x5 protein is one of the putative unknown proteins of sars-cov. the recombinant protein has been successfully expressed in e. coli in the form of insoluble inclusion body. the inclusion body was dissolved in high concentration of urea. affinity chromatography was preformed to purify the denatured protein, and then the product was refolded in a series of gradient solutions of urea. the purified protein was obtained with the purity of > 95% and the yield of 93.3 mg x l(-1). polyclonal antibody of ...200819239038
[effect on mrna and secretion levels of proinflammatory cytokines in dc infected by sars-cov n gene recombinant adenovirus].to investigate the exact mechanism of sars-cov pathogenesis at the protein level.200819544636
detection and phylogenetic analysis of group 1 coronaviruses in south american bats.bat coronaviruses (bt-covs) are thought to be the precursors of severe acute respiratory syndrome coronavirus. we detected bt-covs in 2 bat species from trinidad. phylogenetic analysis of the rna-dependent rna polymerase gene and helicase confirmed them as group 1 coronaviruses.200819046513
signal amplification on a dna-tile-based biosensor with enhanced sensitivity.aims: herein, we report our work to improve the detection sensitivity of a dna-tile-based and self-assembled biosensing platform. this was achieved using hybridization chain reaction (hcr) as a signal amplifier on a water-soluble self-assembled dna nanoarray carrying detection probes. materials & methods: the fluorescence enhancement on the addition of specific detection targets was observed directly by confocal fluorescence microscopy. results & discussion: the versatility of the system was dem ...200818694314
variable oligomerization modes in coronavirus non-structural protein 9.non-structural protein 9 (nsp9) of coronaviruses is believed to bind single-stranded rna in the viral replication complex. the crystal structure of nsp9 of human coronavirus (hcov) 229e reveals a novel disulfide-linked homodimer, which is very different from the previously reported nsp9 dimer of sars coronavirus. in contrast, the structure of the cys69ala mutant of hcov-229e nsp9 shows the same dimer organization as the sars-cov protein. in the crystal, the wild-type hcov-229e protein forms a tr ...200818694760
polymorphisms in the c-type lectin genes cluster in chromosome 19 and predisposition to severe acute respiratory syndrome coronavirus (sars-cov) infection.polymorphisms of clec4m have been associated with predisposition for infection by the severe acute respiratory syndrome coronavirus (sars-cov). dc-signr, a c-type lectin encoded by clec4m, is a receptor for the virus. a variable number tandem repeat (vntr) polymorphism in its neck region was recently associated with susceptibility to sars infection. however, this association was controversial and was not supported by subsequent studies. two explanations may account for this discrepancy: (1) ther ...200818697825
dissection and identification of regions required to form pseudoparticles by the interaction between the nucleocapsid (n) and membrane (m) proteins of sars coronavirus.when expressed in mammalian cells, the nucleocapsid (n) and membrane (m) proteins of the severe acute respiratory syndrome coronavirus (sars-cov) are sufficient to form pseudoparticles. to identify region(s) of the n molecule required for pseudoparticle formation, we performed biochemical analysis of the interaction of n mutants and m in hek293 cells. using a peptide library derived from n, we found that amino acids 101-115 constituted a novel binding site for m. we examined the ability of n mut ...200818703211
molecular dynamic simulations analysis of ritonavir and lopinavir as sars-cov 3cl(pro) inhibitors.since the emergence of the severe acute respiratory syndrome (sars) to date, neither an effective antiviral drug nor a vaccine against sars is available. however, it was found that a mixture of two hiv-1 proteinase inhibitors, lopinavir and ritonavir, exhibited some signs of effectiveness against the sars virus. to understand the fine details of the molecular interactions between these proteinase inhibitors and the sars virus via complexation, molecular dynamics simulations were carried out for ...200818706430
a sars-cov protein, orf-6, induces caspase-3 mediated, er stress and jnk-dependent apoptosis.severe acute respiratory syndrome (sars) coronavirus (cov) spread from china to more than 30 countries, causing severe outbreaks of atypical pneumonia and over 800 deaths worldwide. cov primarily infects the upper respiratory and gastrointestinal tract; however, sars-cov has a unique pathogenesis because it infects both the upper and lower respiratory tracts and leads to human respiratory diseases. sars-cov genome has shown containing 14 open reading frames (orfs) and 8 of them encode novel prot ...200818708124
expression and functional characterisation of the putative sars coronavirus non-structural proteins x1-x5.1. we produced mammalian expression vectors encoding the sars coronavirus (sars-cov) accessory proteins with or without the fluorescence protein tag and cell lines with stable expression of these proteins. 2. the cellular localisation and function of the sars-cov accessory proteins was determined. 3. sars 6 and sars 8b proteins are localised to the endoplasmic reticulum and nucleus/cytoplasm, respectively, and both proteins stimulate host cell dna synthesis.200818708666
sars coronavirus and apoptosis.1. the adenovirus-mediated overexpression of sars coronavirus (sars-cov) spike protein (s) and its c-terminal domain (s2) induce apoptosis in vero e6 cells. 2. such apoptosis in vero e6 cells is time- and dose-dependent. 3. the adenovirus-mediated overexpression of sars-cov n-terminal domain (s1) and other structural proteins, including e,m and n protein, do not induce apoptosis.200818708667
molecular and genetic characterisation of the sars coronavirus auxiliary protein x1 in drosophila.1. we have generated monoclonal antibodies against the sars coronavirus (sars-cov) x1/3a protein (3a), which are suitable for western blotting, immunocytochemistry, and immunohistochemistry. 2. we have established and characterised an in-vivo 3a transgenic drosophila model, and demonstrated its usefulness in studying sars-cov 3a gene function. 3. we validated our in-vivo findings on 3a gene function in mammalian vero e6 cells. 4. our findings raise the possibility of using ion channel blockers a ...200818708668
risk-stratified seroprevalence of sars coronavirus in children residing in a district with point-source outbreak compared to a low-risk area.1. sars coronavirus has low transmissibility at the community level. 2. subclinical sars coronavirus infection is rare in children.200818708669
mechanisms of lymphocyte loss in sars coronavirus infection.1. human lymphocytes and monocytes are not permissive to productive sars coronavirus (sars-cov) infection in vitro. 2. challenge of lymphocytes and monocytes with infectious sars-cov, inactivated virions, and receptor-binding fragment of spike protein does not trigger apoptosis. 3. direct infection/interaction between viruses and lymphocytes/monocytes is unlikely to be the cause of lymphopaenia in sars patients. 4. lymphopaenia in sars patients is likely to result from indirect mechanisms second ...200818708670
investigation of immunogenic t-cell epitopes in sars virus nucleocapsid protein and their role in the prevention and treatment of sars infection.1. a novel hla-a2.1-specific sars coronavirus (sars-cov) nucleocapsid (n) protein epitope (n220-n228 lalllldrl) able to activate cytotoxic t cells in vitro has been identified. 2. when used with a single-chain-trimer system, the sars-cov n protein epitope (n220-n228 lalllldrl) can stimulate a cytotoxic t-cell response against n-protein expressing cells in the hla-a2.1k(b) transgenic mouse model.200818708671
role of polymorphisms of the inflammatory response genes and dc-signr in genetic susceptibility to sars and other infections.1. a genetic risk-association study involving more than 1200 subjects showed individuals homozygous for l-sign tandem repeats are less susceptible to sars infection. 2. this was supported by in vitro binding studies that demonstrated homozygous l-sign, compared to heterozygous, had higher binding capacity for sars coronavirus (sars-cov), with higher proteasome-dependent viral degradation. in contrast, homozygous l-sign demonstrated lower binding capacity for hiv1-gp120.3. genetic-association stu ...200818708672
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