CN1283789C - New Aike 30 Virus and its use - Google Patents
New Aike 30 Virus and its use Download PDFInfo
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- CN1283789C CN1283789C CNB2004100534310A CN200410053431A CN1283789C CN 1283789 C CN1283789 C CN 1283789C CN B2004100534310 A CNB2004100534310 A CN B2004100534310A CN 200410053431 A CN200410053431 A CN 200410053431A CN 1283789 C CN1283789 C CN 1283789C
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Abstract
The present invention belongs to the field of a microbe virus, which particularly relates to a new strain of an Echo30 virus and purposes thereof. By adopting isolated culture of specimens of a patient's cerebrospinal fluid and feces and the neutralization test of an enterovirus and using an RT-PCR technique for sequence and comparison of the gene amplification of viral capsid protein VP1, the present invention proves that the separated virus is an Echo30 mutated strain named Echo30-FDJS03, and the preservation number of the strain is CGMCCNo. 1199. The virus of the present invention is the first nucleic acid sequence information of an Echo30 separating strain in China, replenishes the preservation of the enterovirus seed in China, provides valuable biologic information of the Echo30 virus and has important functions for the diagnosis of diseases caused by the pathogen, the perfection of an infection monitoring system, the popularization control of the diseases, etc. The Echo30-FDJS03 strain can also be used for the research of preparing quick diagnosing reagents and vaccines for Echo30 virus infections.
Description
Technical field
The invention belongs to the microbial virus field, relate to the new strain of virus, be specifically related to new strain that a kind of dust can 30 types (Echo30) virus and uses thereof.
Background technology
It is reported have the aseptic meningitis of clear and definite cause of disease to be caused by enterovirus basically in recent years, wherein Echo30 is one of main pathogen.The outburst of meningitis that Echo30 causes, China from the nineties from the beginning of there being some areas to report successively, but scale is all little, European and American countries has then just had a lot of outbursts reports since the eighties.The separation of Echo30 virus at present mainly relies on cell cultures, and type identifies except traditional neutralization test, and development in recent years is got up is that the various Protocols in Molecular Biologies of basic means also become viral rapid identification method in succession with RT-PCR.Wherein be subjected to more sure be the mensuration of all or part of sequence of virus VP 1.The 1-7 month in 2003, fairly large aseptic meningitis eruption and prevalence has taken place in urban district, Yancheng, China Jiangsu Province and 8 affiliated counties thereof.Totally 1757 people morbidity mainly is the children below 14 years old, and the male sex is more than the women, and sex ratio is about 2: 1.Clinically mainly show as heating, headache, vomiting, intracranial hypertension, stiffness of the neck, individual patient has cough, and symptoms of digestive tract such as diarrhoea are rare; In conjunction with the result of cerebrospinal fluid biochemical analysis and microbial culture feminine gender, clinical diagnosis is a cause of disease aseptic meningitis to be looked into.Because the outburst cause of disease is not clear, can not in time take effective measure of control, caused fairly large disease popularity outburst.
Summary of the invention
The purpose of this invention is to provide a kind of novel dust can 30 types (Echo30) virus stain, strain of the present invention can prepare the Echo30 diagnostic reagent, be used for the on-the-spot quick diagnosis of disease, controlling flow professional etiquette mould is for improving Echo30 and the enterovirus Monitoring systems provides bioinformation.
Another object of the present invention is to adopt new E cho30 virus stain, for the preparation of enterovirus vaccine provides information.
The present invention adopts the cerebrospinal fluid of inpatient and the separation and Culture that stool sample carries out virus, utilize the RT-PCR preliminary evaluation to be enterovirus, carry out the neutralization test of strain then by enterovirus combination serum and the various monovalent antiserum that gets, strain is fixed to Echo30 type enterovirus.Simultaneously, with the back order-checking of increasing of the gene of viral capsid proteins VP1, enter GenBank and search the gene type that strain is carried out in comparison with the RT-PCR technology, confirm isolating virus be the variation strain of Echo30.Called after Echo30-FDJS03-102 strain, its preserving number are CGMCC No.1199, preservation date: on July 28th, 2004.
Specifically, dust provided by the invention can 30 C-type virus C Echo30-FDJS03-102 strains, have the structure and the following characteristics of sequence 1,
(1) cells infected has the tear drop shape cytopathy, comprises that cellular contraction becomes circle, and fragmentation floats in the nutrient solution;
(2) the negative staining electron microscope look is observed, and shows spherical virus particle, meets the known form morphology of virus of Echo virus;
(3) serum neutralization test can neutralize fully virus cytopathic effect;
(4) the VP1 sequence with Echo30 prototype-strain Bastianni has 82% homology.
The present invention is undertaken by following method and step,
1) pathogen isolation is cultivated: according to a conventional method, the samples of CSF direct inoculation is in individual layer MRC-5 cell, ight soil is handled method (the World Health Organization.Specimen receipt and processing.In:Polio Laboratory Manual-underrevision.Geneva:WHO that recommends with reference to WHO, 2001.69-74), with complete PBS stool sample is made 20% suspension, handle through chloroform, 4 ℃ of centrifugal 20min of 1500g, get supernatant and be inoculated in the MRC-5 cell, at 5%CO
2Cultivate under 37 ℃ of conditions, pair cell pathology effect every day (CPE) is observed, and after cultivating 7d, gets supernatant behind the centrifugal 20min of sick cell nutrient solution 3000rmp, and separating viral particles carries out negative staining electron microscope to be observed.
2) neutralization test: on 96 orifice plates, carry out microneutralization test.According to ordinary method, with 50 μ l 100TCID
50Each group (type) serum of enterovirus of virus and equivalent mixes, 37 ℃ of effect 2h get 50 μ l mixtures then and are inoculated in individual layer MRC-5 cell, establish negative cells contrast and positive-virus synchronously and contrast 5%CO
237 ℃ of cultivations, every day the observation of cell pathology, continuous 7 days.
3) RT-PCR and VP1 sequencing: extract viral RNA with the Trizol method; With randomhexamer is primer, and 37 ℃ of effects of MMLV (Promega) 1h is c-DNA with the RNA reverse transcription; The PCR cycling condition of VP1 is: 94 ℃ of 5min; 94 ℃ of 30s, 51 ℃ of 30s, 72 ℃ of 30s, 72 ℃ of 7min.The VP1 amplified production checks order on ABI3730 type dna sequence dna automatic analyser after purifying.
Result's demonstration,
(1) normal MRC-5 cell is a human embryonic lung diploid fibroblast, is fibrous growth; The typical tear drop shape cytopathy of enterovirus occurs behind the positive separation of the cell infection sample, show as cellular contraction and become circle, in addition broken, float in the nutrient solution.
(2) the negative staining electron microscope look is observed morphology of virus, and cells infected is got supernatant and carried out the observation of negative staining electron microscope look after cultivating 7 days behind the culture low-speed centrifugal, show the spherical virus particle of diameter at 20-30nm.The known form that meets Echo virus.
(3) at first make up antiserum(antisera) and (comprise CoxA9, CoxB1～6, Echo1～7,9 with KMB, 11～15,17,19～21,24,25,27,32 totally 28 serotypes) carry out neutralization test, without any one group of serum can be fully in and the cytopathic effect of isolated strain; Then carry out neutralization test with the various monovalent antiserum that is not included in the KMB combination antiserum(antisera), the result shows to have only can neutralize the fully cytopathic effect of virus of Echo30 type monovalent antiserum under test dose.Show that isolated strain is an Echo30 type enterovirus.
(4) strain isolated virus successfully amplifies the 440bp target stripe at the RT-PCR through the guiding of enterovirus universal primer.The a pair of primer amplification that lays respectively at VP3 and 2A district goes out to comprise the purpose fragment of complete VP1 gene (876nt), the purifying order-checking.By the retrieval comparison that blastn and the blastp program of GenBank are carried out close gene, confirm that strain isolated virus is the Echo30 variant that 82% homology is arranged with the VP1 sequence of Echo30 prototype-strain Bastianni.
(5) complete VP1 sequence of the present invention has been submitted GenBank to, and the accession no. of acquisition is AY665609.
The cell that adopts in the inventive method is a MRC-5 cell well known in the art.
The enterovirus combination serum and the various monovalent antiserum that adopt in the inventive method are provided by China Medical Sciences Academy Medical Biology Institute, the primer of required enterovirus universal primer of RT-PCR and amplification VP1 is international endorsement primer sequence (Oberste MS, Maher K, Kennett ML, et al.Molecular epidemiology and genetic diversityof echovirus type 30 (E30): genotypes correlate with temporaldynamics of E30 isolation.J Clin Microbiol, 1999,37:3928-3933), synthetic by living worker in Shanghai and Bo Ya two tame Bioisystech Co., Ltd, the VP1 amplified production is purified and is checked order by rich Asia.
Echo30-FDJS03 strain of the present invention is the domestic nucleic acid sequence information that obtains the Echo30 strain isolated first, and it is committed to GenBank.It is a kind of replenishing that the present invention not only preserves China's enterovirus seed culture of viruses, provide China Echo30 virus extremely valuable bioinformation simultaneously, the medical diagnosis on disease that this cause of disease is caused, monitoring of infection system improve and associated diseases popular control etc. all plays an important role.
Echo30-FDJS03 strain of the present invention also can be used for preparing the fast diagnosis reagent and the vaccine research of Echo30 virus infection.
Description of drawings
Fig. 1 keeps 72 hours normal MRC-5 cells behind the liquid for nutrient solution replaces with.
Fig. 2 is for infecting 10
5TCID
50The MRC-5 cell that virus is back 72 hours.
The spherical virus particle of Fig. 3 negative staining electron microscope look visible 20-30nm.
RT-PCR amplified production electrophoresis photo under the guiding of Fig. 4 440bp enterovirus universal primer.
Fig. 5 part of V P1 sequencing result.
Embodiment
Embodiment 1
Gather urban district, Yancheng, China Jiangsu Province and 8 affiliated counties thereof break out in January, 2003 part meningitis inpatient cerebrospinal fluid and stool sample test.Be separated to virus 22 strains that can produce obvious cytopathy and can continous-stable go down to posterity propagation by cell cultures, be fixed to Echo30.
1) pathogen isolation is cultivated: according to a conventional method, the samples of CSF direct inoculation is in individual layer MRC-5 cell, ight soil is handled the method for recommending with reference to WHO, with complete PBS stool sample is made 20% suspension, handle through chloroform, 4 ℃ of centrifugal 20min of 1500g get supernatant and are inoculated in the MRC-5 cell, at 5%CO
2Cultivate under 37 ℃ of conditions, pair cell pathology effect every day (CPE) is observed, and after cultivating 7d, gets supernatant behind the centrifugal 20min of sick cell nutrient solution 3000rmp, and separating viral particles carries out negative staining electron microscope to be observed.
2) neutralization test: on 96 orifice plates, carry out microneutralization test.According to ordinary method, with 50 μ l 100TCID
50Each group (type) serum of enterovirus of virus and equivalent mixes, 37 ℃ of effect 2h get 50 μ l mixtures then and are inoculated in individual layer MRC-5 cell, establish negative cells contrast and positive-virus synchronously and contrast 5%CO
237 ℃ of cultivations, every day the observation of cell pathology, continuous 7 days.
3) RT-PCR and VP1 sequencing: extract viral RNA with the Trizol method; With randomhexamer is primer, and 37 ℃ of effects of MMLV (Promega) 1h is c-DNA with the RNA reverse transcription; The PCR cycling condition of VP1 is: 94 ℃ of 5min; 94 ℃ of 30s, 51 ℃ of 30s, 72 ℃ of 30s, 72 ℃ of 7min.The VP1 amplified production checks order on ABI3730 type dna sequence dna automatic analyser after purifying.
The result shows that the typical tear drop shape cytopathy of enterovirus appears in the MRC-5 cell that infects virus of the present invention, and cellular contraction becomes circle, even broken, floats in the nutrient solution; The negative staining electron microscope look is observed, and shows the spherical virus particle of diameter at 20-30nm, meets the known form of Echo virus; Neutralization test result shows, has only can neutralize fully under the test dose cytopathic effect of virus of Echo30 type monovalent antiserum, confirms that institute's isolated strain is an Echo30 type enterovirus; By the retrieval comparison that blastn and the blastp program of GenBank are carried out close gene, confirm that institute's strain isolated virus is the Echo30 variant that 82% homology is arranged with the VP1 sequence of Echo30 prototype-strain Bastianni.
SEQUENCE LISTING sequence table
＜110〉Fudan University
＜120〉a kind of dust can 30 C-type virus Cs and uses thereof
<160>1
<170>PatentIn?version?3.2
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<211>876
<212>RNA
<213>Echovirus?30
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<400>1
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uacuuuagac?augugaacaa?aucgacugcu?uacccaguca?auaguguugc?ccgugucuac 720
uuuaaaccca?agcaugucaa?ggccugggug?ccccgcgcgc?cacgcuugug?cccguauuug 780
uaugcaagga?augucaacuu?ugaugugcaa?ggggugaccg?agucucggga?caaaauuacu 840
cuugaccgau?caacccauaa?uccucuguca?aacacu 876
Claims (7)
1, a kind of dust can 30 C-type virus C Echo30-FDJS03-102 strains, and its preserving number is CGMCC No.1199, has the structure and the following characteristics of sequence 1,
(1) cells infected has the tear drop shape cytopathy, comprises that cellular contraction becomes circle, and fragmentation floats in the nutrient solution;
(2) the negative staining electron microscope look is observed, and shows spherical virus particle, meets the known form morphology of virus of Echo virus;
(3) serum neutralization test can neutralize fully virus cytopathic effect;
(4) the VP1 sequence with Echo30 prototype-strain Bastianni has 82% homology.
2, dust according to claim 1 can 30 C-type virus C Echo30-FDJS03-102 strains, it is characterized in that described Echo30-FDJS03-102 strain diameter is 20-30nm.
3, the described dust of claim 1 can the purposes of 30 C-type virus C Echo30-FDJS03-102 strains in preparation enterovirus diagnostic reagent.
4, purposes according to claim 3, wherein said enterovirus are Echo30 virus.
5, the described dust of claim 1 can the purposes of 30 C-type virus C Echo30-FDJS03-102 strains in preparation aseptic meningitis diagnostic reagent.
6, the described dust of claim 1 can the purposes of 30 C-type virus C Echo30-FDJS03-102 strains in the vaccine of preparation Echo30 virus infection.
7, the described dust of claim 1 can the purposes of 30 C-type virus C Echo30-FDJS03-102 strains in preparation aseptic meningitis vaccine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100534310A CN1283789C (en) | 2004-08-04 | 2004-08-04 | New Aike 30 Virus and its use |
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| CNB2004100534310A CN1283789C (en) | 2004-08-04 | 2004-08-04 | New Aike 30 Virus and its use |
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| CN1283789C true CN1283789C (en) | 2006-11-08 |
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Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111153989B (en) * | 2020-01-15 | 2020-09-15 | 江苏省疾病预防控制中心(江苏省公共卫生研究院) | Preparation and application of Echo30 monoclonal antibody |
| CN111153990B (en) * | 2020-01-15 | 2020-10-27 | 江苏省疾病预防控制中心(江苏省公共卫生研究院) | Monoclonal antibody aiming at Echo30, preparation method and application thereof |
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