CN1468955A - New type of coronavirus - Google Patents
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Abstract
The present invention discloses one new kind of coronavirus, which is circular, has coronary fiber lugs arranged in the peripheral, has diameter of 80-120 nm and is distributed in kytoplsm. The virus is obtained through separating sample of SARS and may be used to inoculate sensitive cell to obtain CPE. The virus can produce specific reaction to serum of clinically diagnosed SARS patient and is one kind of SARS virus.
Description
Technical field
The present invention relates to a kind of new virus, relate in particular to a kind of novel coronavirus that causes that serious acute respiratory system synthesis is levied.
Background technology
Severe acute respiratory system synthesis levy (Severe acute respiratory syndrome, SARS) be recently in the Asia, a kind of acute respiratory transmissible disease of occurring of North America and Europe, the domestic atypical pneumonia that is referred to as.This disease is close contact and the short range contagiousness droplet infection by the person to person mainly since this disease onset anxious, propagate fast, lethality rate is higher, brings very big threat for the people's health, and society stable also caused certain impact.Finding out the infective pathogen body of atypical pneumonia, is to carry out targetedly the basis of diagnosing, preventing and treating.In order to find out the SARS pathogenic agent, work out effectively preventing method and measure of control, the countries in the world scientist unites tackling key problem, seeks the SARS cause of disease hand in hand.The expert of China Sickness Prevention Control Center Virus Disease Prevention Control Institute at first finds chlamydozoan sample particle by Electronic Speculum in patient becomes celestial sample, think that chlamydozoan may be the cause of disease of atypical pneumonia; The scientist in Hong Kong once thought in early days in research and was likely the current atypical pneumonia that influenza or avian influenza virus mutation cause; On March 19th, 2003 (DOH such as Manuel Dayrit, SARS virus may belong to family ofmicrobes causing measles and mumps.http: //www.doh.gov.ph/sars/measles_and_mumps.htm/2003-03-19) sample to severe respiration syndrome case carries out thinking after the preliminary study that the pathogenic agent of SARS may belong to the paramyxovirus coe virus.The scientist of Hong Kong University's department of microbiology announces to turn out coronavirus from the SARS sample after a few days, thinks that coronavirus may be the pathogenic agent of SARS.
Summary of the invention
The invention discloses a kind of novel coronavirus, this virus belongs to coronaviridae (Coronaviridae), and coronavirus genus (Coronavirus) is sars coronavirus.Four strains of this virus are respectively from area, Guangzhou and Beijing area atypical pneumonia death lung tissue sample, separate in Beijing area atypical pneumonia patient Nasopharyngeal swabs sample and Beijing area atypical pneumonia death liver and the lymphoglandula mixed structure sample, difference called after GZ01, BJ01, BJ02, the BJ03 strain, in on June 5th, 2003 in (No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City, China Committee for Culture Collection of Microorganisms common micro-organisms center, Institute of Microorganism, Academia Sinica) preservation, the preservation registration number of four strain virus is respectively CGMCC NO.0939, CGMCC NO.0936, CGMCC NO.0937, CGMCC NO.0938.
How spherical in shape this virion is or oval, and periphery has the fibre of crown arrangement prominent, and it is spherical that lug tips is, the fine gap that broad is arranged between prominent, viral diameter between 80～120nm, fine prominent long 20～40nm.See Fig. 2 a.In addition, also see rod-short, kidney shape and difform virion, long 120-220nm, wide 50-90nm, fine prominent long 20～40nm, single or double, be petal-shaped.This virion mainly is distributed in the cytoplasmic endoplasmic reticulum cisterna of cells infected, golgi body, the cavity and the extracellular, also sees the virion that is dispersed in sometimes in kytoplasm.Sophisticated virion periphery is double membrane structure clearly as seen.Virion is hollow and solid two states, and the virion of same shape often comes across in the same viral inclusion body, also can see fine prominent virion at inclusion body that has and extracellular.It is spherical or oval that viral inclusion body mostly is, and its form, difference in size are bigger, and the inclusion body inner virus is arranged closely.Can see that virus enters in the cell with pinocytosis or film fusion form, it is gemmation that virus discharges the extracellular.The cell mitochondrial swelling of infective virus, part mitochondrial outer membrane and ridge dissolving, reticulum dilatation, nucleus chromatin condensation, limit collection.
The 4 strain virus genom sequences of coronavirus of the present invention are respectively AY278489 in the accession number of America NI H GenBank, AY278488, AY278487 and AY278490.
Sequential analysis shows that the homology of virus strain of the present invention and known person and ani mal coronavirus is all less than 70%.Phylogenetic tree figure shows isolating virus, no matter is and human corona virus or all far away with the sibship of ani mal coronavirus.Therefore, isolating virus be a kind of new, coronavirus of having morphed.
The present inventor proves that by seroepidemiological survey and neutralization test virus of the present invention is in close relations with present popular atypical pneumonia, is the main pathogens that causes current popular atypical pneumonia.
The contriver utilizes novel coronavirus to infect suckling mouse, cause erratic morbidity of suckling mouse and death, and from the infected mice lung preparation, observe the coronavirus particle, in conjunction with the isolation identification of novel coronavirus, the serological research of pathogen relation, prove that fully the pathogenic agent that causes this atypical pneumonia is a novel coronavirus.
The present invention has following method to the isolation identification of pathogenic agent:
1, sample disposal
Tissue of patient sample and blood preparation: get the lung tissue sample that becomes celestial and be ground into homogenate, add an amount of viral dilution liquid, get supernatant after centrifugal and be used for inoculating cell training and animal; The Nasopharyngeal swabs sample: microbiotic is added in the sample, and 4 ℃ of backs of spending the night are centrifugal, and supernatant liquor is used for inoculation.
2, pathogen isolation is cultivated
Suckling mouse inoculation method: under aseptic condition,, if morbidity, then get suckling mouse brain, lung tissue grinds to form suspension, inoculation suckling mouse, 3 generations of blind passage continuously with microsyringe suckling mouse intracranial inoculation or abdominal cavity inoculation simultaneously, observation incidence.
Cell culture method: with the specimen inoculation Tissue Culture Flask of handling well, observation of cell changes, if CPE not occurring does not carry out blind passage, and 3 generations of blind passage continuously.
3, electron microscopic morphology is observed
The negative staining electron microscope look is checked: collect the cell culture fluid that the bacterial isolate body infects, and fixing, negative staining, transmission electron microscope observing.Ultrastructure is checked: gets the cell of infection, fixes through glutaraldehyde, and the isotonic buffer solution washing, osmic acid is fixed, gradient ethanol dehydration, embedding, conventional ultrathin section(ing), the two dyeing of acetic acid uranium-lead citrate, transmission electron microscope observing.
4, indirect IF staining
The preparation of isolate cells infected sheet: with cell inoculation isolate cell culture fluid, cultivate the absorption back on slide.Treat that pathology appears in cell, cultivate and make cell attachment.Not attached cell is removed in rinsing, and fixing seal is preserved behind the airing.Indirect immunofluorescence antibody dyeing process: with patients serum's sample dilution deactivation, suitably be added drop-wise on the cell antigen sheet after the dilution, 37 ℃ of effect post-flush airings add goat-anti people FITC mark fluorescent antibody more again, effect post-flush airing in 37 ℃ of wet boxes, the fluorescence microscope coloration result.
5, micro-cell cultures neutralization test (fixed virus-dilute serum antibody act)
Get coronavirus nutrient solution liquid, measure TCID50, with atypical pneumonia patient antibody positive serum and normal controls serum dilution back deactivation, hybrid virus and serum are put in 37 ℃ of incubators and after the effect virus-serum mixture inoculation sensitive cells are cultivated.Calculate half protective number (PD
50).
6, RT-PCR amplification
Prepare total RNA in the sample to be checked, carry out the reverse transcription PCR amplification, the agarose electrophoresis observations.
7, sequencing, homology comparison and evolutionary tree are analyzed
To check order behind the RT-PCR product purification, carry out the Blast homology relatively, according to the known coronavirus gene order of Genbank registration, use the MegAlign module of DNAstar software, employing JotunHein method carries out the virus corresponding sequence comparison of many canopies shape and drawing system is set.
Virus of the present invention can be used for the diagnostic reagent of SARS, the aspects such as preparation of anti-SARS vaccin.Applicant of the present invention has adopted this virus to prepare coronavirus antibody detection kit (number of patent application is 03124299.5).
Description of drawings
Fig. 1 a is normal Vero-E6 cell.
Fig. 1 b is 3 days a Vero-E6 cell of lung tissue sample's inoculation.
Fig. 2 a is become celestial sars coronavirus particle in the lung tissue sample inoculation Vero-E6 cell culture fluid of Beijing area death, negative staining, transmission electron microscope.
Fig. 2 b is that area, Guangzhou sample infects the virion in the suckling mouse lung tissue, ultrathin section(ing).
Fig. 2 c is become celestial virion in lung tissue sample's vero cells infection of Beijing area death.
Fig. 2 d is become celestial a virion in lung tissue sample's vero cells infection of Guangzhou area death.
Fig. 3 is atypical pneumonia patients serum and isolating virus immunity fluorescent dye reaction.
Fig. 4 is the new phylogenetic analysis figure that separates coronavirus based on the portion gene sequence.
Embodiment
Embodiment one causes isolation identification one, the material of SARS pathogenic agent
1, sample: 2 parts of lung tissue samples, respectively from area, Guangzhou and Beijing area " atypical pneumonia " death; The Nasopharyngeal swabs sample picks up from Beijing area " atypical pneumonia " patient in hospital.
2, serum specimen: atypical pneumonia patients serum, accept the people that diagnoses a disease really for medical treatment from Guangzhou and Beijing area hospital; Normal human serum picks up from Beijing and Guangzhou area blood station blood donor.
Clones such as 3, cell: Vero-E6, MDCK, Hep-2, Hela, BHK-21 and LLC-MK-2 are commercial cell strain.
4, substratum: RPIM 1640, DMEM, Gibco/BRL company product; Calf serum: Hangzhou Sijiqing Biological Engineering Material Co., Ltd.'s product; Penicillin, Streptomycin sulphate: be North China Pharmaceutical Factory's product.
5, laboratory animal: BALB/C and Kunming kind small white mouse suckling mouse: the 3-5 age in days is the court's Experimental Animal Center breeding supply.
6, RT-PCR and amplified production purified reagent, primer: be used for the RNeasy MiniKit of RNA preparation, purchase company in QIAGEN; ThermoScript II SuperScript II, the Taq archaeal dna polymerase is available from Invitrogen company; The nucleic acid gel reclaims test kit QIAquick Gel Extraction Kit available from QIAGEN company, and primer is synthetic by Beijing three rich polygala root biotech companies.Two, method
1, sample disposal
Tissue of patient sample: get the lung tissue sample that becomes celestial, in the mortar of precooling, be ground into homogenate, add an amount of viral dilution liquid (1640 substratum that contain 500u/ml penicillin, 500u/ml Streptomycin sulphate, pH7.0), the centrifugal 20min of 3000rpm gets supernatant and is used for inoculating cell training and animal.Blood preparation: the patient blood grumeleuse grinds to form homogenate in the mortar of precooling, adds an amount of viral dilution liquid, makes it to grind to form the suspension of 10-20%, and the centrifugal 20min of 2000rpm, supernatant liquor are used for inoculation; The Nasopharyngeal swabs sample: press in 1000u/ml penicillin, the 1000 μ g/ml Streptomycin sulphates adding sample, 4 ℃ are spent the night, and the centrifugal 30min of 3000rpm, supernatant liquor are used for inoculation.
2, pathogen isolation is cultivated
The suckling mouse inoculation method: select healthy 3-5 age in days suckling mouse, under aseptic condition, with the microsyringe intracranial inoculation of 0.25mL, every duplicate samples inoculation a brood of (at least 5), every suckling mouse intracranial inoculation 0.02mL, or the 0.03mL/ of abdominal cavity inoculation simultaneously is only.Observe the incidence of mouse every day, overview 7-14 days.If the suckling mouse morbidity occurs, suckling mouse is dissected got brain, lung tissue in observation period, grind to form 1 0% tissue suspension, inoculation suckling mouse, 3 generations of blind passage continuously.
Cell culture method: with the specimen inoculation handled well in the 10mL Tissue Culture Flask that grows up to monolayer cell, two bottles of every part of specimen inoculation, 37 ℃ of absorption 1h, add the cell maintenance medium that contains 2% calf serum at sucking-off specimen fluids then, put 37 ℃ of CO2 incubators and cultivate.Every day, observation of cell changed, and observed continuously 10 days.If CPE do not occur and do not carry out blind passage, 3 generations of blind passage continuously.
3, electron microscopic morphology is observed
The negative staining electron microscope look is checked: collect the Vero-E6 cell culture fluid that the bacterial isolate body infects, with 6.1%, after glutaraldehyde stationary liquid and the nutrient solution of pH 7.2 fix by 1: 1 mixed, with 3% Salkowski's solution negative staining, transmission electron microscope observing.Ultrastructure is checked: get the Vero-E6 cell that infects suckling mouse lung tissue, lung tissue sample and become celestial lung tissue sample's infection, with 3.1%, pH 7.2 glutaraldehyde fix, 1/15molL, pH7.2 phosphoric acid salt isotonic buffer solution washing 3 times, after 1% osmic acid is fixing, the gradient ethanol dehydration, the Epon812 embedding, conventional ultrathin section(ing), the two dyeing of acetic acid uranium-lead citrate, transmission electron microscope observing.
4, indirect IF staining
The preparation of isolate cells infected sheet: VERO-E6 cell Hank ' the s liquid that will cultivate into individual layer is washed once, inoculates isolate cell cultures suspension then, and the 1mL/ bottle is put in 37 ℃ of incubators and adsorbed 1h, adds cell maintenance medium, puts 37 ℃ and continues to cultivate.When treating that cell just pathology occurred, get cells infected and drip sheet, put 37 ℃ of CO dripping good slide
2Continue to cultivate 8h in the incubator and make cell attachment.Take out slide, put into 0.005M pH7.2 PBS rinsing, remove not adherent suspension cell, airing.Put into cold acetone ,-20 ℃ of fixing 30min, airing ,-20 ℃ of sealings are preserved standby.
The indirect immunofluorescence antibody dyeing process: after the dilution in 1: 10 of patients serum's sample, 56 ℃ of effect 30min, and then suitably be added drop-wise to respectively on the cell antigen sheet after the dilution, put and act on 30min (then acting on 90min) in 37 ℃ of wet boxes if detect IgM antibody, flushing, airing is washed in the PBS vibration 3 times.Add goat-anti people FITC mark fluorescent antibody then, put in 37 ℃ of wet boxes and act on 30min, flushing is the same, and airing is observed coloration result under fluorescent microscope.
5, micro-cell cultures neutralization test (fixed virus-dilute serum antibody act)
Get test with newly separating crown virus-culturing fluid liquid, determine the TCID of viral liquid
50Titre, standby.Atypical pneumonia patient antibody positive serum and normal controls serum are diluted the back in 56 ℃, 30min deactivation at 1: 5.
TCID according to the viral suspension of measuring
50Titre is diluted virus with viral dilution liquid (DMEM+2% calf serum), is diluted to 400 TCID
50/ 100 μ L viral suspensions, standby.Simultaneously, the patients serum and the control serum of deactivation carried out serial doubling dilution, promptly get 0.1mL serum and add isopyknic physiological saline, serial dilution successively with physiological saline.
At first, hybrid virus and serum are got different each 0.15mL of dilution serum respectively, with 400TCID
50/ 100 μ L viral suspensions add respectively in the serum tube of dilution, and 0.15mL/ pipe makes and respectively manages virus concentration and be 200 TCID
50/ 100 μ L fully act on 1h in the rearmounted 37 ℃ of incubators of mixing.
Virus-serum mixture inoculation sensitive cells is inoculated into virus-serum mixture in the VERO-E6 cell that microtest plate cultivates, and each concentration is inoculated 4 holes, and cell maintenance medium 100 μ L are added in 50 μ L/ holes again, puts into 37 ℃ and contains the 5%CO2 incubator and cultivate.
Establish virus control simultaneously: viral dilution is become 2000,200,20,2 TCID
50/ 100 μ L, each extent of dilution inoculate 4 cell cultures holes, and every hole adds viral suspension 50 μ l; Negative serum contrast: except that replace the test serum other all same test group with negative serum; The normal cell contrast: establish 4 hole normal cell contrasts, every hole adds diluent 50 μ L.
Day by day observation of cell pathology, and record.Cultivate and changed cell maintenance medium once in 3-4 days.Observe a week.
Half protective number (PD50) calculates: according to the cytopathy situation, calculate in 50% serum and terminal point by the Reed-Muench method.Earlier calculate distance proportion by following formula; distance proportion=(mortality ratio of 50%-＜50%)/(mortality ratio of＞50% mortality ratio-＜50%); again logarithm (the Log)+distance proportion of＜50% mortality ratio serum dilution is taken advantage of the logarithm of dilution factor, promptly draw half protective number (PD50).
6, RT-PCR amplification
The preparation of total RNA in the sample to be checked, press RNeasy Mini Kit test kit specification sheets, by atypical pneumonia patient's internal organs mixtures such as the lung tissue that becomes celestial, infecting mouse lung tissue and liver spleen thereof, and extract total RNA in the Vero E-6 cell of Nasopharyngeal swabs inoculation and the culture supernatant thereof.
At first carry out reverse transcription, in pipe, add the total RNA of 5 μ L, 1 μ L Superscript II ThermoScript II (200u), 1 μ L primer 4Bm2 (or CV2,3), 4 μ l, 5 * reaction buffer, 1 μ L RNA enzyme inhibitors and 1 μ L 10mM dNTP and 2 μ L 0.1M DTT supply volume to 20 μ L with the water that does not contain RNase at last.Behind 42 ℃ of reaction 60min, 95 ℃ of deactivation 5min.Carry out pcr amplification then, reactive system comprises: 5 μ l, 10 * PCR reaction buffer, 1 μ L 10mM dNTP, 2 μ L25mM MgCl
2, 1 μ L TaqDNA polysaccharase (2.5u), each 1 μ L (10 μ M) of upstream and downstream primer, water complements to 50 μ L.With 2Bp/4Bm, CV1/CV2 and BLF+/BLF-primer to 40 and 35 circulations of increasing respectively.Reaction finishes, with product through 1% agarose gel electrophoresis observations.
7, sequencing, homology comparison and evolutionary tree are analyzed
Behind the RT-PCR product purification, check order by instrument room, BIO ENGINEERING INST MILITARY center; Also can be with the fragment cloning of purifying to the T-easy carrier, the screening positive clone order-checking.The row that check order carry out the Blast homology relatively, present known coronavirus gene order according to the Genbank registration, use the MegAlign module of DNAstar software, employing Jotun Hein method carries out the virus corresponding sequence comparison of many canopies shape and drawing system is set.Three, result
1, pathogen isolation is cultivated
Cell cultures is separated: selected 5 strain passage cell strains inoculation case samples such as Vero-E6, MDCK, Hep-2, Hela and BHK-21, carried out the atypical pneumonia pathogen isolation.Isolate pathogenic agent the Nasopharyngeal swabs sample that utilizes lung tissue that Vero-E6, mdck cell become celestial from area, Guangzhou and Beijing area respectively and Beijing area to gather, see Table 1.Tangible cytopathy (CPE) occurs in 3 days after the specimen inoculation Vero-E6 cell cultures, see Fig. 1 a, Fig. 1 b, and can stablize and go down to posterity, the stable isolate that passed for 7 generations is preserved as seed culture of viruses.On mdck cell, also observed tangible CPE, but the time that occurs is later, needs 5 days.Fail to isolate pathogenic agent with other 3 strain cells.In addition, also with the isolating pathogenic agent of Vero-E6 inoculation LLC-MK-2 cell, isolating pathogenic agent also can be grown on the LLC-MK-2 cell for we, but the time that CPE occurs will arrive and inoculates back 7 days.
Table 1, cell cultures bacterial isolate body sample Vero-E6 MDCK Hep-2 Hela BHK-211++---2++---3++---No. 1 as a result are area, Guangzhou the dead lung tissue that becomes celestial; Be Beijing area the dead lung tissue that becomes celestial No. 2; No. 3 is Patients in Beijing Area Nasopharyngeal swabs sample.
Among the result the present inventor separate four canopy shape virus stains: GZ01 strain and BJ01 strain, separate respectively from Guangzhou area and Beijing area atypical pneumonia death lung tissue sample; The BJ02 strain separates from Beijing area atypical pneumonia patient Nasopharyngeal swabs sample; The BJ03 strain separates from the Beijing area " atypical pneumonia " death liver and lymphoglandula mixed structure sample.
Utilize suckling mouse to separate and utilize suckling mouse to carry out pathogen isolation, the first-generation has suckling mouse morbidity, death after the intracranial inoculation, but it is irregular to fall ill.The mouse brain of getting morbidity continues to go down to posterity, and suckling mouse still can fall ill erratically at present.
2, the electron microscopic morphology of pathogenic agent is observed
The negative staining electron microscope look is checked: Guangzhou and Beijing area become celestial in the Vero-E6 cell culture fluid of lung tissue sample's inoculation, and negative staining is all looked into and seen virion, and is how rounded, and periphery has the fibre of crown arrangement prominent, and viral diameter is seen Fig. 2 a between 80-120nm.
Ultrastructure is checked: look in the suckling mouse pulmonary epithelial cells of Guangzhou blood preparation inoculation morbidity and see that virion, virus are dispersed in and be distributed in the cytoplasm, rounded, diameter is many about 90nm, sees Fig. 2 b.The cell mitochondrial swelling of infective virus, part mitochondrial outer membrane or ridge dissolving.All look in the Vero-E6 cell that the lung tissue sample that becomes celestial regional in Guangzhou and the Beijing area infects and see a large amount of virions, how rounded, diameter is about 80nm.Virion mainly is distributed in the endoplasmic reticulum cisterna, endochylema cavity of endochylema and Fig. 2 c, Fig. 2 d are seen, the integrated heap of poly in the extracellular.The visible reticulum dilatation of the cell of infective virus, mitochondrial swelling, ridge dissolving, nucleus chromatin condensation, limit collection.
3, the serology of blood samples of patients sample detects
In order to confirm the relation of isolating pathogenic agent and popular atypical pneumonia, we are prepared into cells infected antigen sheet with isolating virus infection Vero-E6 cell, in order to detect the antibody that whether has anti-isolating virus among the patients serum.Gather 26 parts of patients serums that clinical diagnosis is an atypical pneumonia from Beijing and area, Guangzhou, had the antibody of anti-isolating virus in 23 parts of patients serum's samples of indirect IF staining proof, seen Fig. 3.And 30 parts of normal human serum total negatives.Positive rate of antibody is 88.5%, and the virus of display separation is in close relations with current popular atypical pneumonia.
4, RT-PCR amplification and sequential analysis
From the lung tissue sample that becomes celestial of Guangzhou and Pekinese's two routine atypical pneumonia cases in the cell culture of isolating pathogenic infection, degenerated primer (2Bp/4Bm) [Poutanen SM, LowDE, Henry B with coronavirus genus, Finkelstein S, Rose D, Green K, Tellier R, Draker R, Adachi D, AyersM, Chan AK, Skowronski DM, Salit I, Simor AE, Slutsky AS, Doyle PW, Krajden M, PetricM, Brunham RC, and McGeer AJ.Ident ification of severe acute respiratory syndromein Canada.N Engl J Med.Low-1 ~ low-11 (at www.nejm.org on March 31,2003)], can obtain the DNA band of a single about 250bp by RT-PCR amplification, its size with expect consistent.Measurement result to this amplified fragments nucleotide sequence shows that itself and human corona virus OC43 type homology are 67%, is respectively 70% and 69% with the homology of avian infectious segmental bronchus coronavirus and mouse pneumonia coronavirus.
Primer [Stephensen CB with other two pairs of coronavirus pol gene conserved regions, CaseBolt DB, and Gangopadhyay NN.Phylogenetic analysis of a highly conserved region of thepolymerase gene from 11 coronaviruses and development of a consensus polymerasechain reaction assay.Virus Res.1999,60:181-189], same amplifying from isolating pathogenic agent and the dna fragmentation of expecting that size is identical, length is respectively 289bp and 207bp.Wherein human corona virus's homology is 53.3% in 289bp fragment and the coronaviridae, and with the bovine coronavirus homology be 67.3%; 207bp fragment and human corona virus's homology are 53.6%, and with the murine hepatitis virus homology be 60%.Result to its sequencing shows, this section sequence of isolating pathogenic agent is identical in Guangzhou and Beijing atypical pneumonia case lung tissue, and the prompting two places cause that the atypical pneumonia popular may be identical virus strain.
Meanwhile, adopt above-mentioned primer directly to carry out RT-PCR amplification, can amplify expection size and the identical dna fragmentation of nucleotide sequence equally from the two routine atypical pneumonias lung tissue that becomes celestial.The result shows, our isolating pathogenic agent is really from the lung tissue that becomes celestial.From the pathogenic agent of the Vero E-6 cellular segregation of Beijing area atypical pneumonia patient Nasopharyngeal swabs inoculation, adopt above-mentioned primer, amplify dna fragmentation corresponding, that size is all identical with sequence equally respectively.
According to present registered coronavirus gene order among the Genbank, adopt Jotun Hein method drawing system generation tree graph, show isolating virus, no matter be and human corona virus or all far away with the sibship of ani mal coronavirus, its homology all is no more than 70%, sees Fig. 4.Therefore, isolating virus may be a kind of coronavirus new, that morphed.The 4 strain virus genom sequences of coronavirus of the present invention are respectively AY278489 in the accession number of America NI H GenBank, AY278488, AY278487 and AY278490.
5, the pathogen relation of isolating novel coronavirus and popular atypical pneumonia
Geographic two tame hospitals and Beijing area four tame hospitals collect 113 parts of the patients serums that clinical diagnosis is an atypical pneumonia altogether from Guangzhou, and wherein the area, Guangzhou is 21 parts, 92 parts of Beijing areas.Utilization is prepared into cell-virus antigen sheet by the isolated 4 strain novel coronavirus of cell cultures, and the antibody by the anti-new isolating coronavirus in the indirect immunofluorescence detection patient serum the results are shown in Table 2 among the patients serum.
Table 2, IFA method are to Beijing-4 30 27 3 90.0, Beijing-3 972 77.8, Beijing-2 14 10 4 71.4, Beijing-1 39 39 0 100, Guangzhou-2 14 11 3 78.6, clinical Patients with Severe Acute Respiratory Syndrome serum IgG antibody testing result hospital positive negative positive rate (%) Guangzhou-1 752 71.4 of numbering detection number of samples sum 113 99 14 87.6
Detect 99 parts of antibody that contain anti-novel coronavirus from patients serum's sample of 113 parts of clinical definites, serum antibody total positives rate is 87.6%.The positive rate that detects of each hospital's sample has certain difference, and high reaches 100%, and low also reaches 71.4%.84 portions of normal human serums have been detected simultaneously, wherein 30 parts of Beijing areas, 24 parts in area, Guangzhou, 30 parts in other area, total negative as a result.These results show that the isolating coronavirus that makes new advances is closely related with current popular atypical pneumonia.
Find simultaneously, and though in the sample of Beijing isolating novel coronavirus strain (BJ03) still isolating strain (GZ01) in the sample of Guangzhou all can produce good antigen-antibody reaction with these serum for BJ01, BJ02.
The anti-new variation that separates coronavirus antibody among the patients serum
Successively collected 10 atypical pneumonia patients' paired sera, wherein Beijing area and Guangzhou are geographic each 5.First part of serum was gathered in morbidity in 5-12 days, and second part of serum 1-3 week after first part of serum collection gathers.The paired sera antibody test the results are shown in Table 3.
Table 3. patient paired sera IFA detects antibody changing conditions patient's blood sampling time antibody titer antibody rising sequence number (morbidity fate) (multiple) 181: 20
25 1∶2560 1282 12 1∶40
28 1∶2560 643 10 1∶320
26 1∶2560 84 7 1∶20
24 1∶320 165 10 1∶160
24 1∶2560 166 5 1∶320
23 1∶1280 47 7 1∶320
20 1∶2560 88 12 1∶20
25 1∶160 89 6 1∶20
11 1∶160 810 6 1∶80
14 1∶640 8
In certain phase, along with the prolongation of patient's disease time, paired sera antibody all has tangible rising, and the highest rising reaches 128 times, 4 times of low also risings.The rising of serum antibody proves that these patients have been novel coronavirus in the recent period and have infected.
Patients serum's antibody is to the neutralizing effect of new separation coronavirus
Select the anti-new isolating coronavirus antibody male atypical pneumonia patients serum of indirect immunofluorescence assay, geographic each 2 parts of Beijing area and Guangzhou, with the BJ01 strain virus is representative, takes the method for fixed virus-dilute serum that new isolating coronavirus is carried out neutralization test.The result proves, 4 parts of serum
Strain all has neutralizing effect to novel coronavirus BJ01.Calculate according to the half protective number calculation formula of introducing in the method, the half protective number of 4 parts of serum is respectively: No. 1 serum in Beijing area 1: 160; No. 2 serum in Beijing area 1: 452; No. 1 serum in area, Guangzhou 1: 768; No. 2 serum in area, Guangzhou 1: 783.These results demonstrate the Beijing area and the geographic patients serum in Guangzhou has stronger neutralization activity to the BJ01 strain virus, show the virus stain that causes this two places atypical pneumonia in and do not have too big difference on the antigen site.
By serological method, verify the etiology relation of a certain pathogenic agent and corresponding transmissible disease, be one of important means of a kind of new infectious disease pathogens of conclusive evidence.The present invention by to the mensuration of anti-new isolating coronavirus antibody among the atypical pneumonia patients serum of clinical definite, comparison that paired sera antibody changes and patients serum to the observation of novel coronavirus neutralizing effect, proved that new isolating coronavirus is the pathogenic agent of this popular atypical pneumonia.
Claims (2)
1, a kind of novel coronavirus is respectively sars coronavirus BJ01 CGMCC NO.0936, sars coronavirus BJ02 CGMCC NO.0937, sars coronavirus BJ03 CGMCCNO.0938, sars coronavirus GZ01 CGMCC NO.0939.
2, the application of the described virus of claim 1 in preparation SARS diagnostic reagent, vaccine.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004085633A1 (en) * | 2003-03-24 | 2004-10-07 | The University Of Hong Kong | A novel human virus causing severe acute respiratory syndrome (sars) and uses thereof |
| CN100413537C (en) * | 2004-01-29 | 2008-08-27 | 国家人类基因组南方研究中心 | SARS coronary virus full virus vaccine |
| CN111363847A (en) * | 2020-02-12 | 2020-07-03 | 广州微远基因科技有限公司 | 2019-nCoV rapid detection primer group based on CRISPR technology and application thereof |
| CN113292649A (en) * | 2020-02-24 | 2021-08-24 | 中国科学院微生物研究所 | Human monoclonal antibodies to novel coronaviruses and uses thereof |
| CN113292650A (en) * | 2020-02-24 | 2021-08-24 | 中国科学院微生物研究所 | Human monoclonal antibodies to novel coronaviruses and uses thereof |
-
2003
- 2003-06-12 CN CNB031379664A patent/CN1176210C/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004085633A1 (en) * | 2003-03-24 | 2004-10-07 | The University Of Hong Kong | A novel human virus causing severe acute respiratory syndrome (sars) and uses thereof |
| CN100413537C (en) * | 2004-01-29 | 2008-08-27 | 国家人类基因组南方研究中心 | SARS coronary virus full virus vaccine |
| CN111363847A (en) * | 2020-02-12 | 2020-07-03 | 广州微远基因科技有限公司 | 2019-nCoV rapid detection primer group based on CRISPR technology and application thereof |
| CN111363847B (en) * | 2020-02-12 | 2021-06-29 | 广州微远医疗器械有限公司 | 2019-nCoV rapid detection primer group based on CRISPR technology and application thereof |
| CN113292649A (en) * | 2020-02-24 | 2021-08-24 | 中国科学院微生物研究所 | Human monoclonal antibodies to novel coronaviruses and uses thereof |
| CN113292650A (en) * | 2020-02-24 | 2021-08-24 | 中国科学院微生物研究所 | Human monoclonal antibodies to novel coronaviruses and uses thereof |
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| CN1176210C (en) | 2004-11-17 |
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