Purification and biophysical properties of human coronavirus 229E
Identifieur interne : 000475 ( Istex/Corpus ); précédent : 000474; suivant : 000476Purification and biophysical properties of human coronavirus 229E
Auteurs : John C. HierholzerSource :
- Virology [ 0042-6822 ] ; 1976.
English descriptors
- Teeft :
- Absorption maxima, Academic press, Amino, Amino acids, Biophysical, Biophysical properties, Block titrations, Bromelin digestion, Carbohydrate, Coronavirus, Coronaviruses, Crude tissue culture proteins, Discontinuous tris buffer system, Dowdle, Electron microscopy, Encephalomyocarditis virus, Erythrocyte, Extinction coefficient, Extracellular virus, Glycoprotein, Growth characteristics, Growth curves, Helf, Helf cells, Helf microcultures, Helf monolayers, Hierholzer, Host cell proteins, Human coronavirus, Indirect hemagglutination, Infectious virus, Infectivity, Intracellular virus, Kaye, Light path, Molecular weight, Molecular weights, Neonatal dogs, Normal concentrations, Obijeski, Peak titers, Peak virus production, Phosphate buffer, Plaque titer, Plaque titers, Polypeptide, Precipitin lines, Protein composition, Rabbit antiserum, Radioactive virus, Radiolabeled virus, Respiratory illness, Same time, Sedimentation coefficient, Spectrophotometric scans, Spike proteins, Spinco rotor, Structural proteins, Sucrose, Sucrose gradients, Supernatant fluid, Titer, Total protein, Tris buffer system, Tube titer, Viral, Viral protein, Viral proteins, Virion, Virology, Virus, Virus bands, Virus preparations, Virus purification.
Abstract
Abstract: Coronavirus 229E was grown to high titers in diploid fibroblast cells under medium containing twice the normal concentrations of amino acids and vitamins. Growth curves showed maximum virus production at multiplicities of infection of 0.1 and 1; maximum titers of intracellular virus occurred at 22–24 hr and of extracellular virus at 26 hr postadsorption. Tube infectivity titers ranged from 109.0–109.5 TCID50/ml and plaque titers from 1010.2–1010.9 y PFU/ml at the time of peak virus production, when no cytopathology was evident. Virus titer dropped rapidly between 26 and 56 hr, coincident with increasing cytopathology. A single precipitin band was observed in immunodiffusion and immunoelectrophoresis between concentrated virus preparations and antiserum to purified 229E. Neuraminidase and hemagglutinin assays were negative. Virus was purified by two procedures: adsorption to and elution from human “0” erythrocytes and CaHPO4 gel followed by equilibrium sucrose gradient centrifugation, and PEG precipitation followed by equilibrium glycerol/tartrate gradients and rate zonal sucrose or glycerol/tartrate gradients. Final lots of purified virus containing <0.02% of the crude tissue culture proteins had absorption maxima at 256 nm and minima at 241.2 nm and a mean extinction coefficient of E1cm1% = 54.3 at 256 nm. The fully corrected sedimentation coefficient for the intact virion was S20,v0 = 381 S. PAGE by different techniques revealed seven polypeptides of mean apparent molecular weights between 16,900 and 196,100. Six contained carbohydrate and one contained lipid. Electropherograms of 3H- and 14C-labeled virus were identical to those of stained gels. Two glycoproteins constituting 25% of the virion protein were identified by bromelin digestion as the spike proteins. The density in sucrose and in potassium tartrate was 1.18 g/ml for the virion and 1.15 g/ml for the “despiked” particle.
Url:
DOI: 10.1016/0042-6822(76)90014-3
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Growth curves showed maximum virus production at multiplicities of infection of 0.1 and 1; maximum titers of intracellular virus occurred at 22–24 hr and of extracellular virus at 26 hr postadsorption. Tube infectivity titers ranged from 109.0–109.5 TCID50/ml and plaque titers from 1010.2–1010.9 y PFU/ml at the time of peak virus production, when no cytopathology was evident. Virus titer dropped rapidly between 26 and 56 hr, coincident with increasing cytopathology. A single precipitin band was observed in immunodiffusion and immunoelectrophoresis between concentrated virus preparations and antiserum to purified 229E. Neuraminidase and hemagglutinin assays were negative. Virus was purified by two procedures: adsorption to and elution from human “0” erythrocytes and CaHPO4 gel followed by equilibrium sucrose gradient centrifugation, and PEG precipitation followed by equilibrium glycerol/tartrate gradients and rate zonal sucrose or glycerol/tartrate gradients. Final lots of purified virus containing <0.02% of the crude tissue culture proteins had absorption maxima at 256 nm and minima at 241.2 nm and a mean extinction coefficient of E1cm1% = 54.3 at 256 nm. The fully corrected sedimentation coefficient for the intact virion was S20,v0 = 381 S. PAGE by different techniques revealed seven polypeptides of mean apparent molecular weights between 16,900 and 196,100. Six contained carbohydrate and one contained lipid. Electropherograms of 3H- and 14C-labeled virus were identical to those of stained gels. Two glycoproteins constituting 25% of the virion protein were identified by bromelin digestion as the spike proteins. 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Tube infectivity titers ranged from 109.0–109.5 TCID50/ml and plaque titers from 1010.2–1010.9 y PFU/ml at the time of peak virus production, when no cytopathology was evident. Virus titer dropped rapidly between 26 and 56 hr, coincident with increasing cytopathology. A single precipitin band was observed in immunodiffusion and immunoelectrophoresis between concentrated virus preparations and antiserum to purified 229E. Neuraminidase and hemagglutinin assays were negative. Virus was purified by two procedures: adsorption to and elution from human “0” erythrocytes and CaHPO4 gel followed by equilibrium sucrose gradient centrifugation, and PEG precipitation followed by equilibrium glycerol/tartrate gradients and rate zonal sucrose or glycerol/tartrate gradients. Final lots of purified virus containing >0.02% of the crude tissue culture proteins had absorption maxima at 256 nm and minima at 241.2 nm and a mean extinction coefficient of E1cm1% = 54.3 at 256 nm. The fully corrected sedimentation coefficient for the intact virion was S20,v0 = 381 S. PAGE by different techniques revealed seven polypeptides of mean apparent molecular weights between 16,900 and 196,100. Six contained carbohydrate and one contained lipid. Electropherograms of 3H- and 14C-labeled virus were identical to those of stained gels. Two glycoproteins constituting 25% of the virion protein were identified by bromelin digestion as the spike proteins. 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Growth curves showed maximum virus production at multiplicities of infection of 0.1 and 1; maximum titers of intracellular virus occurred at 22–24 hr and of extracellular virus at 26 hr postadsorption. Tube infectivity titers ranged from 109.0–109.5 TCID50/ml and plaque titers from 1010.2–1010.9 y PFU/ml at the time of peak virus production, when no cytopathology was evident. Virus titer dropped rapidly between 26 and 56 hr, coincident with increasing cytopathology. A single precipitin band was observed in immunodiffusion and immunoelectrophoresis between concentrated virus preparations and antiserum to purified 229E. Neuraminidase and hemagglutinin assays were negative. Virus was purified by two procedures: adsorption to and elution from human “0” erythrocytes and CaHPO4 gel followed by equilibrium sucrose gradient centrifugation, and PEG precipitation followed by equilibrium glycerol/tartrate gradients and rate zonal sucrose or glycerol/tartrate gradients. Final lots of purified virus containing <0.02% of the crude tissue culture proteins had absorption maxima at 256 nm and minima at 241.2 nm and a mean extinction coefficient of E1cm1% = 54.3 at 256 nm. The fully corrected sedimentation coefficient for the intact virion was S20,v0 = 381 S. PAGE by different techniques revealed seven polypeptides of mean apparent molecular weights between 16,900 and 196,100. Six contained carbohydrate and one contained lipid. Electropherograms of 3H- and 14C-labeled virus were identical to those of stained gels. Two glycoproteins constituting 25% of the virion protein were identified by bromelin digestion as the spike proteins. The density in sucrose and in potassium tartrate was 1.18 g/ml for the virion and 1.15 g/ml for the “despiked” particle.</p></abstract></profileDesc><revisionDesc><change when="1976">Published</change></revisionDesc></teiHeader></istex:fulltextTEI><json:item><extension>txt</extension><original>false</original><mimetype>text/plain</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-7GJ05MLP-W/fulltext.txt</uri></json:item></fulltext><metadata><istex:metadataXml wicri:clean="Elsevier, elements deleted: tail"><istex:xmlDeclaration>version="1.0" encoding="utf-8"</istex:xmlDeclaration><istex:docType PUBLIC="-//ES//DTD journal article DTD version 4.5.2//EN//XML" URI="art452.dtd" name="istex:docType"></istex:docType><istex:document><converted-article version="4.5.2" docsubtype="fla"><item-info><jid>YVIRO</jid><aid>76900143</aid><ce:pii>0042-6822(76)90014-3</ce:pii><ce:doi>10.1016/0042-6822(76)90014-3</ce:doi><ce:copyright type="unknown" year="1976"></ce:copyright></item-info><head><ce:title>Purification and biophysical properties of human coronavirus 229E</ce:title><ce:author-group><ce:author><ce:given-name>John C.</ce:given-name><ce:surname>Hierholzer</ce:surname></ce:author><ce:affiliation><ce:textfn>Respiratory Virology Branch, Virology Division, Bureau of Laboratories, Center for Disease Control, Public Health Service, Department of Health, Education and Welfare, Atlanta, Georgia 30333, USA</ce:textfn></ce:affiliation></ce:author-group><ce:date-accepted day="9" month="7" year="1976"></ce:date-accepted><ce:abstract><ce:section-title>Abstract</ce:section-title><ce:abstract-sec><ce:simple-para>Coronavirus 229E was grown to high titers in diploid fibroblast cells under medium containing twice the normal concentrations of amino acids and vitamins. Growth curves showed maximum virus production at multiplicities of infection of 0.1 and 1; maximum titers of intracellular virus occurred at 22–24 hr and of extracellular virus at 26 hr postadsorption. Tube infectivity titers ranged from 10<ce:sup>9.0</ce:sup>–10<ce:sup>9.5</ce:sup> TCID<ce:inf>50</ce:inf>/ml and plaque titers from 10<ce:sup>10.2</ce:sup>–10<ce:sup>10.9</ce:sup> y PFU/ml at the time of peak virus production, when no cytopathology was evident. Virus titer dropped rapidly between 26 and 56 hr, coincident with increasing cytopathology. A single precipitin band was observed in immunodiffusion and immunoelectrophoresis between concentrated virus preparations and antiserum to purified 229E. Neuraminidase and hemagglutinin assays were negative. Virus was purified by two procedures: adsorption to and elution from human “0” erythrocytes and CaHPO<ce:inf>4</ce:inf> gel followed by equilibrium sucrose gradient centrifugation, and PEG precipitation followed by equilibrium glycerol/tartrate gradients and rate zonal sucrose or glycerol/tartrate gradients. Final lots of purified virus containing <0.02% of the crude tissue culture proteins had absorption maxima at 256 nm and minima at 241.2 nm and a mean extinction coefficient of <ce:italic>E</ce:italic><ce:inf>1cm</ce:inf><ce:sup>1%</ce:sup> = 54.3 at 256 nm. The fully corrected sedimentation coefficient for the intact virion was <ce:italic>S</ce:italic><ce:inf>20,v</ce:inf><ce:sup>0</ce:sup> = 381 S. PAGE by different techniques revealed seven polypeptides of mean apparent molecular weights between 16,900 and 196,100. Six contained carbohydrate and one contained lipid. Electropherograms of <ce:sup loc="pre">3</ce:sup>H- and <ce:sup loc="pre">14</ce:sup>C-labeled virus were identical to those of stained gels. Two glycoproteins constituting 25% of the virion protein were identified by bromelin digestion as the spike proteins. The density in sucrose and in potassium tartrate was 1.18 g/ml for the virion and 1.15 g/ml for the “despiked” particle.</ce:simple-para></ce:abstract-sec></ce:abstract></head></converted-article></istex:document></istex:metadataXml><mods version="3.6"><titleInfo><title>Purification and biophysical properties of human coronavirus 229E</title></titleInfo><titleInfo type="alternative" contentType="CDATA"><title>Purification and biophysical properties of human coronavirus 229E</title></titleInfo><name type="personal"><namePart type="given">John C.</namePart><namePart type="family">Hierholzer</namePart><affiliation>Respiratory Virology Branch, Virology Division, Bureau of Laboratories, Center for Disease Control, Public Health Service, Department of Health, Education and Welfare, Atlanta, Georgia 30333, USA</affiliation><role><roleTerm type="text">author</roleTerm></role></name><typeOfResource>text</typeOfResource><genre type="research-article" displayLabel="Full-length article" authority="ISTEX" authorityURI="https://content-type.data.istex.fr" valueURI="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</genre><originInfo><publisher>ELSEVIER</publisher><dateIssued encoding="w3cdtf">1976</dateIssued><copyrightDate encoding="w3cdtf">1976</copyrightDate></originInfo><language><languageTerm type="code" authority="iso639-2b">eng</languageTerm><languageTerm type="code" authority="rfc3066">en</languageTerm></language><abstract lang="en">Abstract: Coronavirus 229E was grown to high titers in diploid fibroblast cells under medium containing twice the normal concentrations of amino acids and vitamins. Growth curves showed maximum virus production at multiplicities of infection of 0.1 and 1; maximum titers of intracellular virus occurred at 22–24 hr and of extracellular virus at 26 hr postadsorption. Tube infectivity titers ranged from 109.0–109.5 TCID50/ml and plaque titers from 1010.2–1010.9 y PFU/ml at the time of peak virus production, when no cytopathology was evident. Virus titer dropped rapidly between 26 and 56 hr, coincident with increasing cytopathology. A single precipitin band was observed in immunodiffusion and immunoelectrophoresis between concentrated virus preparations and antiserum to purified 229E. Neuraminidase and hemagglutinin assays were negative. Virus was purified by two procedures: adsorption to and elution from human “0” erythrocytes and CaHPO4 gel followed by equilibrium sucrose gradient centrifugation, and PEG precipitation followed by equilibrium glycerol/tartrate gradients and rate zonal sucrose or glycerol/tartrate gradients. Final lots of purified virus containing <0.02% of the crude tissue culture proteins had absorption maxima at 256 nm and minima at 241.2 nm and a mean extinction coefficient of E1cm1% = 54.3 at 256 nm. The fully corrected sedimentation coefficient for the intact virion was S20,v0 = 381 S. PAGE by different techniques revealed seven polypeptides of mean apparent molecular weights between 16,900 and 196,100. Six contained carbohydrate and one contained lipid. Electropherograms of 3H- and 14C-labeled virus were identical to those of stained gels. Two glycoproteins constituting 25% of the virion protein were identified by bromelin digestion as the spike proteins. The density in sucrose and in potassium tartrate was 1.18 g/ml for the virion and 1.15 g/ml for the “despiked” particle.</abstract><relatedItem type="host"><titleInfo><title>Virology</title></titleInfo><titleInfo type="abbreviated"><title>YVIRO</title></titleInfo><genre type="journal" authority="ISTEX" authorityURI="https://publication-type.data.istex.fr" valueURI="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</genre><originInfo><publisher>ELSEVIER</publisher><dateIssued encoding="w3cdtf">1976</dateIssued></originInfo><identifier type="ISSN">0042-6822</identifier><identifier type="PII">S0042-6822(00)X0296-6</identifier><part><date>1976</date><detail type="volume"><number>75</number><caption>vol.</caption></detail><detail type="issue"><number>1</number><caption>no.</caption></detail><extent unit="issue-pages"><start>1</start><end>261</end></extent><extent unit="pages"><start>155</start><end>165</end></extent></part></relatedItem><identifier type="istex">2ADBD3AFE80E3A14C8BC39E26B426C6B09BF6F6D</identifier><identifier type="ark">ark:/67375/6H6-7GJ05MLP-W</identifier><identifier type="DOI">10.1016/0042-6822(76)90014-3</identifier><identifier type="PII">0042-6822(76)90014-3</identifier><recordInfo><recordContentSource authority="ISTEX" authorityURI="https://loaded-corpus.data.istex.fr" valueURI="https://loaded-corpus.data.istex.fr/ark:/67375/XBH-HKKZVM7B-M">elsevier</recordContentSource></recordInfo></mods><json:item><extension>json</extension><original>false</original><mimetype>application/json</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-7GJ05MLP-W/record.json</uri></json:item></metadata></istex></record>Pour manipuler ce document sous Unix (Dilib)
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