CN103571799B - One strain duck source H4N6 subtype avian influenza virus strain and application thereof - Google Patents
One strain duck source H4N6 subtype avian influenza virus strain and application thereof Download PDFInfo
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Abstract
The invention discloses strain duck source H4N6 subtype avian influenza virus strain and an application thereof. Does is the preserving number of described virus stain CGMCC? NO.7606. This Strain is duck source H4N6 subtype avian influenza virus, contain CDS sequence as SEQ? ID? NO.1～SEQ? ID? 8 fragments shown in NO.8, correspond respectively to PB2, PB1, PA, HA, NP, NA, M and NS. The present invention has carried out whole genome sequence mensuration to this Strain, and its information is used for studying detection, distribution, the regularty of epidemic of avian influenza virus and controls the popular of bird flu and spread. Strain of the present invention has been supplemented the extremely valuable hereditary information of China H4N6 subtype avian influenza virus, for molecular evolution and the epidemiological study of H4N6 subtype avian influenza virus provide valuable data, fast diagnosis reagent and the vaccine research that can also infect for the preparation of H4N6 subtype avian influenza virus.
Description
Technical field
The invention belongs to animal virology field, relate to the new strain of virus and application thereof that a strain separates, be specially a strainDuck source H4N6 subtype avian influenza virus strain and application thereof.
Background technology
Bird flu (Avianinfluenza, AI) is a kind of serious harm avian health of being caused by A type influenza virusCommunicable disease. Bird infects after avian influenza virus (Avianinfluenzavirus, AIV), and symptom can show as non-dominantInfect, Asia faces and examines infection, or slight breathing problem, egg production reduce until various ways such as acute whole body fatal diseases.AIV is according to its pathogenic highly pathogenic AIV (HighlyPathogenicAvianInfluenzavirus, HPAIV) that is divided intoWith low pathogenicity AIV (LowPathogenicAvianInfluenzavirus, LPAIV). H4 hypotype belongs to low pathogenicityHow AIV, be separated in aquatic bird and wild bird body, sustainable existence in the body such as duck, goose, and may propagate to chicken. Although theseVirus is low pathogenicity to animal body more, but can make egg production decline, thereby causes damage to aviculture. As far back as 1956,Czechoslovakia is just in duck and has been separated to H4N6, and 1999, Alexander reported first suffered from pneumonia from CanadaIn pig body, be separated to H4N6 hypotype AIV, and also have similar report in Russia and China, show that this subtype virus also can be in kindBetween propagate. The host range of H4 hypotype AIV is wider, comprises chicken, turkey, duck, wild duck, ostrich, parrot, dead seal and sick pigOn be also once separated to H4 hypotype AIV, illustrate that H4 hypotype AIV also has mammal certain pathogenic. In addition, H4 hypotypeAIV may infect H5, H9 or other hypotypes AIV after infecting again, and in body, its NA gene and internal gene may occur multipleAssorted genetic recombination, produces novel recombinant virus. Therefore,, in AI monitoring, also should cause to H4 hypotype AIV the pass of heightNote. AIV exists antigenic drift and antigenic shift phenomenon widely, and its antigenic variation frequency is very high. Therefore, if can be to AIVCarry out whole genome sequence mensuration, for studying detection, distribution, the regularty of epidemic of avian influenza virus and controlling the stream of bird fluRow, spread all tool is had very great significance.
Summary of the invention
Technical problem to be solved by this invention is to exist antigenic drift and antigenic shift phenomenon widely for AIV,The present situation that its antigenic variation frequency is very high, and a strain duck source H4N6 subtype avian influenza virus strain is carried out to whole genome sequence surveyFixed, and its information is used for studying detection, distribution, the regularty of epidemic of avian influenza virus and controls the popular and climing of bird fluProlong. The genomic information of H4N6 subtype avian influenza virus strain of the present invention has great value.
One of technical scheme provided by the invention is: a strain influenza A virus strain, its preserving number is CGMCCNO.7606。
Described influenza A virus strain is duck source H4N6 subtype avian influenza virus strain A/duck/Shanghai/Y20/06 (H4N6), near the amino acid sequence viral HA gene cleavage site is " PEKASR ↓ GLF ", is low pathogenicity fowlInfluenza strain feature. Described influenza A virus strain is preserved in Chinese microorganism strain preservation pipe on May 13rd, 2013Reason committee common micro-organisms center (CGMCC), address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, the Chinese Academy of Sciences is micro-Biological study institute, its Classification And Nomenclature is: influenza A virus, its deposit number is CGMCCNO.7606.
Described influenza A virus strain, the CDS order of its PB2, PB1, PA, HA, NP, NA, a M and NS8 genetic fragmentRow are respectively as shown in SEQIDNO:1～SEQIDNO:8. Wherein HA and NA gene and H4N6 subtype avian influenza virus are in sameIn one branch; And PB2, PB1, NP, PA gene are with at present in close relations at the popular H6 subtype avian influenza virus of China; M geneWith A/environment/Korea/CSM05/2004 (H3N1) in same branch; And NS gene and A/wildduck/Korea/YS44/2004 (H1N2) homology is the highest, and therefore, this is a strain recombinant fowl influenza virus strain.
Two of technical scheme provided by the invention is: the influenza A virus poison that aforementioned preserving number is CGMCCNO.7606The preparation method of strain, it comprises step: the influenza A virus strain that is CGMCCNO.7606 by preserving number is with 0.2mL/ pieceThe SPF chicken embryo of inoculum concentration inoculation 9～10 ages in days, the effective dose of wherein said strain is 104～105EID50/ mL, then will connectChicken embryo after kind is placed in incubator and cultivates 48～96h and discard chicken embryo dead in 24h, collects the allantoic fluid of infected chicken embryo.
Three of technical scheme provided by the invention is: the influenza A virus poison that aforementioned preserving number is CGMCCNO.7606The application of strain in preparation H4N6 subtype influenza virus diagnose reagent.
Four of technical scheme provided by the invention is: the influenza A virus poison that aforementioned preserving number is CGMCCNO.7606The application of strain in preparation H4 subtype avian influenza virus vaccine.
Five of technical scheme provided by the invention is: a kind of nucleic acid of separation, its nucleotide sequence as SEQIDNO.1,SEQIDNO.2, SEQIDNO.3, SEQIDNO.4, SEQIDNO.5, SEQIDNO.6, SEQIDNO.7 and SEQShown in any in IDNO.8.
The nucleic acid of described nucleotide sequence as shown in SEQIDNO.1 is that aforementioned preserving number is CGMCCNO.7606'sThe CDS sequence of the PB2 genetic fragment of influenza A virus strain;
The nucleic acid of described nucleotide sequence as shown in SEQIDNO.2 is that aforementioned preserving number is CGMCCNO.7606'sThe CDS sequence of the PB1 genetic fragment of influenza A virus strain;
The nucleic acid of described nucleotide sequence as shown in SEQIDNO.3 is that aforementioned preserving number is CGMCCNO.7606'sThe CDS sequence of the PA genetic fragment of influenza A virus strain;
The nucleic acid of described nucleotide sequence as shown in SEQIDNO.4 is that aforementioned preserving number is CGMCCNO.7606'sThe CDS sequence of the HA genetic fragment of influenza A virus strain;
The nucleic acid of described nucleotide sequence as shown in SEQIDNO.5 is that aforementioned preserving number is CGMCCNO.7606'sThe CDS sequence of the NP genetic fragment of influenza A virus strain;
The nucleic acid of described nucleotide sequence as shown in SEQIDNO.6 is that aforementioned preserving number is CGMCCNO.7606'sThe CDS sequence of the NA genetic fragment of influenza A virus strain;
The nucleic acid of described nucleotide sequence as shown in SEQIDNO.7 is that aforementioned preserving number is CGMCCNO.7606'sThe CDS sequence of the M genetic fragment of influenza A virus strain;
The nucleic acid of described nucleotide sequence as shown in SEQIDNO.8 is that aforementioned preserving number is CGMCCNO.7606'sThe CDS sequence of the NS genetic fragment of influenza A virus strain.
Meeting on the basis of this area general knowledge, above-mentioned each optimum condition, can be combined, and obtains respectively better reality of the present inventionExample.
Agents useful for same of the present invention and raw material be commercially available obtaining all.
Positive progressive effect of the present invention is: the duck source H4N6 subtype avian influenza disease that the invention provides a kind of restructuringPoison. This virus contains 8 fragments of CDS sequence as shown in SEQIDNO.1～SEQIDNO.8, correspond respectively to PB2,PB1, PA, HA, NP, NA, M and NS. Strain of the present invention has been supplemented the extremely valuable of China H4N6 subtype avian influenza virusHereditary information, for molecular evolution and the epidemiological study of H4N6 subtype avian influenza virus provide valuable data, can also be used forFast diagnosis reagent and vaccine research that preparation H4N6 subtype avian influenza virus infects.
Strain of the present invention is measured and gene evolution relationship analysis through whole genome sequence, shows that this virus is a strainThe strain being developed through complicated restructuring in duck body by the gene in the different subtype sources such as H4N6, H6N2, H6N5, H3N1 and H1N2Recombinant virus. Therefore, to the understanding of H4N6 subtype avian influenza virus genetic evolution information, heavy to grasping between influenza virus hostGroup, trace to the source, enrich virus base, the monitoring of related diseases substance is significant.
Biomaterial preservation information
A/duck/Shanghai/Y20/06 provided by the invention (H4N6) Strain, in preservation on May 13 in 2013At China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), preservation address: the Chaoyang District, Beijing City North StarNo. 3, No. 1, West Road institute, postcode: 100101. The deposit number of this germ strain is: CGMCCNo.7606.
Brief description of the drawings
Fig. 1 is the systematic evolution tree of HA gene;
Fig. 2 is the systematic evolution tree of NA gene;
Fig. 3 is the systematic evolution tree of PB2 gene;
Fig. 4 is the systematic evolution tree of PB1 gene;
Fig. 5 is the systematic evolution tree of PA gene;
Fig. 6 is the systematic evolution tree of NP gene;
Fig. 7 is the systematic evolution tree of M gene;
Fig. 8 is the systematic evolution tree of NS gene.
Detailed description of the invention
Mode below by embodiment further illustrates the present invention, but does not therefore limit the present invention to described realityExecute among routine scope. The experimental technique of unreceipted actual conditions in the following example, according to conventional method and condition, or according to businessProduct description is selected.
In following embodiment, the primer and probe are synthetic by the English Weihe River prompt base (Shanghai) trade Co., Ltd.
Separation and the preservation of embodiment 1H4N6 subtype avian influenza virus
1, the separation of H4N6 subtype avian influenza virus
Collection duck field, China District of Shanghai healthy duck 20 parts of larynxes, cloaca cottons are wiped and separate culture identification. WarpChicken embryo culture is separated to virus 1 strain of energy aggegation chicken red blood cell, is accredited as H4N6 subtype avian influenza virus, specific as follows.
(1) virus separates and cultivates: collection duck larynx, cloaca cotton are wiped, and are kept in PBS buffer solution, and in 4h, cold chain transportsTo laboratory. Duck larynx, cloaca cotton are wiped sample treatment and cultivate the method with reference to GB/T18936-2003, and sample is through penicillin(3000IU/mL), streptomysin (3mg/mL) processes 1h in 4 DEG C, cotton is wiped fully after twisting, wringing out and discards swab, sample liquid warpThe centrifugal 10min of 1000r/min, gets supernatant and (tests purchased from Cimmeria dimension in Beijing is logical through allantoic cavity inoculation 9～10 age in days SPF chicken embryosAnimal Technology Co., Ltd.), 0.2mL/ piece, wherein virus load is about 104～105EID50/ mL, put and in incubator, cultivate 48～96h, observes 2 every day, discards chicken embryo dead in 24h, collects the allantoic fluid of infected chicken embryo. In GB/T19438.1-2004Described method is accredited as avian influenza virus.
(2) H4N6 subtype avian influenza virus genome sequencing:
Adopt general the drawing of the amplification avian influenza virus pnca gene of sequence as shown in SEQIDNO.9～SEQIDNO.23Thing (HOFFMANNE, STECHJ, GUANY, etal.Universalprimersetforthefull-lengthamplificationofallinfluenzaviruses[J].ArchivesofVirology,2001,146(12):8 genetic fragments of Strain 2275-2289) (1) being obtained are carried out RT-PCR amplification, obtain size and are about respectively8 fragments of 2.3kb, 2.3kb, 2.2kb, 1.7kb, 1.5kb, 1.4kb, 1.0kb and 0.9kb, correspond respectively to PB2, PB1,PA, HA, NP, NA, a M and NS8 genetic fragment, all with expection in the same size, wherein M is formed by connecting by M2 and M1, NS have NS2 andNS1 is formed by connecting. Shown in universal primer used is specific as follows:
Ba-PB2-1:TATTGGTCTCAGGGAGCGAAAGCAGGTC；
Ba-PB2-2341R:ATATGGTCTCGTATTAGTAGAAACAAGGTCGTTT(corresponding to SEQIDNO.9 andSEQIDNO.10, for the PB2 that increases).
Bm-PB1-1:TATTCGTCTCAGGGAGCGAAAGCAGGTAC；
Bm-PB1-2341R:ATATCGTCTCGTATTATTAGAAACAAGGCATTT(corresponding to SEQIDNO.11 andSEQIDNO.12, for the PB1 that increases).
Bm-PA-1:TATTCGTCTCAGGGAGCGAAAGCAGGTAC；
Bm-PA-2233R:ATATCGTCTCGTATTATTAGAAACAAGGTACTT(is corresponding to SEQIDNO.13 and SEQIDNO.14, for the PA that increases).
Bm-HA-1:TATTGGTCTCAGGGAGCGAAAGCAGGGG；
Bm-NS-890R:ATATCGTCTCGTATTATTAGAAACAAGGGTGTTTT(corresponding to SEQIDNO.15 andSEQIDNO.16, for the HA that increases).
Bm-NP-1:TATTGGTCTCAGGGAGCGAAAGCAGGGTA；
Bm-NP-1565R:ATATCGTCTCGTATTATTAGAAACAAGGGTATTTTT(corresponding to SEQIDNO.17 andSEQIDNO.18, for the NP that increases).
Bm-NA-1:TATTGGTCTCAGGGAGCGAAAGCAGGAGT；
Bm-NA-1413R:ATATCGTCTCGTATTATTAGAAACAAGGAGTTTTTT(corresponding to SEQIDNO.19 andSEQIDNO.20, for the NA that increases).
Bm-M-1:TATTGGTCTCAGGGAGCGAAAGCAGGTAG；
Bm-M-1027R:ATATCGTCTCGTATTATTAGAAACAAGGTAGTTTTT(corresponding to SEQIDNO.21 andSEQIDNO.22, for the M that increases).
Bm-NS-1:TATTGGTCTCAGGGAGCGAAAGCAGGGTG；
Bm-NS-890R:ATATCGTCTCGTATTATTAGAAACAAGGGTGTTTT(corresponding to SEQIDNO.23 andSEQIDNO.16, for the NS that increases).
Concrete operations are first the chick embryo allantoic liquid of infection Invitrogen company's T rizolLSReagent reagent to be taken outCarry viral nucleic acid, AMV reverse transcriptase (Promega) reverse transcription, reverse transcription condition is 42 DEG C, 1h; 95 DEG C, 5min. Then to contraryThe cDNA that transcribes acquisition utilizes PremixLATaq(TaKaRa) carry out pcr amplification, PCR condition is: 94 DEG C, 3min; 30 are followedRing (94 DEG C, 20s; 58 DEG C, 30s; 72 DEG C, 7min); 72 DEG C, 10min. The product finally pcr amplification being obtained is served seaInvitrogen company carries out sequencing.
Result demonstration, this Strain is H4N6 subtype avian influenza virus; Full gene to virus of the present invention carries out sequence surveyDetermine and adopt MEGA4.0 analysis software to carry out homology and evolutionary analysis (seeing Fig. 1～8). H4N6 influenza virus A of the present invention/Duck/Shanghai/Y20/06 (H4N6) has 8 fragments, and complete genome sequence and the feature of each fragment are specific as follows:
1) HA fragment complete genome sequence and isolate A/duck/Taiwan/5910/2006 (H4N6) 98.8% homology;
2) NA fragment complete genome sequence and isolate A/mallard/Yanchen/2005 (H4N6) 98.3% homology;
3) PB1 fragment complete genome sequence and isolate A/mallard/Jiangxi/7376/2003 (H6N2) 95.7% is sameSource;
4) PB2 fragment complete genome sequence and isolate A/duck/EasternChina/31/2004 (H6N2) 98.9% is sameSource;
5) PA fragment complete genome sequence and isolate A/duck/Hokkaido/120/2001 (H6N2) 98.1% homology;
6) M fragment complete genome sequence and isolate A/environment/Korea/CSM05/2004 (H3N1) 99.7% is sameSource;
7) NP fragment complete genome sequence and isolate A/duck/Hunan/5613/2003 (H6N5) 99.0% homology;
8) NS fragment complete genome sequence and isolate A/wildduck/Korea/YS44/2004 (H1N2) 99.3% homology.
Can learn the nucleotides of institute's isolates HA gene and A/duck/Taiwan/5910/2006 (H4N6) from resultSequence has 98.8% homology; NA gene and A/mallard/Yanchen/2005 (H4N6) in same branch, nucleotidesHomology is 98.3%; And PB2, PB1, NP, PA gene are with at present in close relations at the popular H6 subtype avian influenza virus of China; MGene and A/environment/Korea/CSM05/2004 (H3N1) are in same branch; And NS gene and A/wildduck/Korea/YS44/2004 (H1N2) homology is the highest. And 8 genes of A/duck/Shanghai/Y20/06 and H4N6 Asia, AmericaType AIV separated strain is not all in same genetic evolution branch, and genetic affinity is far away each other. Above result shows, A/Duck/Shanghai/Y20/06 may be that gene by the different subtype such as H4N6, H6N2, H6N5, H3N1 and H1N2 source is duckThe strain recombinant virus developing through complicated restructuring in body.
2, the preservation of H4N6 subtype avian influenza virus
This viral whole genome sequence (CDS sequence) as shown in SEQIDNO.1～SEQIDNO.8, wherein its HAWith NA gene and H4N6 subtype avian influenza virus in same branch; And PB2, PB1, NP, PA gene with flow in China at presentThe H6 subtype avian influenza virus of row is in close relations; M gene and A/environment/Korea/CSM05/2004 (H3N1) inSame branch; And NS gene and A/wildduck/Korea/YS44/2004 (H1N2) homology is the highest, therefore, this is a strainRecombinant strain. Near amino acid sequence this virus HA gene cleavage site is " PEKASR ↓ GLF ", is low pathogenicity bird fluStrain feature. Called after A/duck/Shanghai/Y20/06 (H4N6), depositary institution's title: Chinese microorganism strain preservation pipeReason committee common micro-organisms center, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Chinese Academy of Sciences's microbe researchInstitute, preservation date: on May 13rd, 2013, preserving number is CGMCCNO.7606, and Classification And Nomenclature is: influenza A virus.
The preparation of embodiment 2 virus stains
The fowl influenza virus strain that is CGMCCNO.7606 by preserving number is with 0.2mL/ pieces (104～105EID50/ mL)Inoculum concentration inoculation 9～10 age in days SPF chicken embryos, are placed in incubator by postvaccinal chicken embryo and cultivate 48～96h and discard in 24h deadThe chicken embryo of dying, collects the allantoic fluid of infected chicken embryo and get final product. The condition of cultivating is:
The preparation of embodiment 3 vaccines
By Strain of the present invention for the preparation of H4N6 subtype avian influenza virus vaccine.
1, the preparation of vaccine
(1) by the centrifugal 10min of DK/SH/Y20/2006 strain virus liquid 5000 × g of preparation, get supernatant.
(2) in supernatant, add formaldehyde, the volumn concentration that makes formaldehyde is that 0.1%, 37 DEG C of deactivation 24 hours is (in bottleTemperature reaches 37 DEG C and starts timing), jolting during this time 3～4 times, obtains inactivation of viruses liquid. Get inactivation of viruses liquid, inoculate 9～10Age, SPF chicken embryo, for subsequent use after the assay was approved.
(3) emulsification
Oil phase preparation: 94 parts by volume injection white oils (purchased from Hangzhou Refinery) and 6 parts by volume Jia Siben-80(are purchased from upperThe popular medicine company in sea), fully mix, (purchased from Shanghai oceangoing voyage chemical reagent work, every 100 milliliters of mixed liquors add then to add aluminum stearate1.75 grams of aluminum stearates), be stirred to gradually transparent till, autoclaving is for subsequent use.
Water preparation: get 3 parts by volume sterilizing Tween-80s, add 97 parts by volume inactivation of viruses liquid, shake well, make tween-80 dissolve completely.
Emulsification: get oil phase 3 parts by volume and be put in emulsion tank, start the motor stirring that slowly runs, slowly add water 1 simultaneouslyParts by volume, stirs 10～30 minutes with 10000r/min after adding again, makes water-in-oil emulsion, before termination is stirred, adds 1%The thimerosal aqueous solution, making its final concentration is 0.01%, makes oil emulsion inactivated vaccine.
Inoculate after vaccine provided by the invention, can greatly increase the prevention ability of chick to avian influenza virus, greatly carryThe economic benefit of high aquaculture industry, has great value for the control of avian influenza virus.
The preparation of embodiment 4 diagnostic reagents
Antigen: described duck source H4N6 fowl influenza virus strain, it suppresses experiment as H4N6 hypotype AIV blood clotting and blood clotting(HA-HI) use antigen.
Positive serum: with embodiment 3 prepare and the chicken at 1 monthly age of oil-emulsion inactivated vaccine immunity (anti-without H4N6 hypotype AIVBody), in isolated rearing device, raise preparation H4 hypotype positive serum, the conventional water feeding of feeding between feeding period. Immunity twice altogether,Between interval 2 weeks, immunity is for the second time collected blood in 14 days, prepares respectively positive serum.
H4 hypotype AIV antigen provided by the invention and positive serum can be for the qualifications of H4 hypotype AIV, for AIV'sClassification diagnosis is significant. In use, suppressing experiment (HA-HI) by AIV blood clotting and blood clotting can realize virusDiagnosis.
Claims (5)
1. a strain influenza A virus strain, is characterized in that, its preserving number is CGMCCNO.7606.
2. the preparation method of the influenza A virus strain that preserving number is CGMCCNO.7606, is characterized in that, it comprises stepRapid: the influenza A virus strain that is CGMCCNO.7606 by preserving number is inoculated 9～10 ages in days with the inoculum concentration of 0.2mL/ pieceSPF chicken embryo, the effective dose of wherein said strain is 104～105EID50/mL, then postvaccinal chicken embryo is placed in to incubatorMiddle cultivation 48～96h also discards chicken embryo dead in 24h, collects the allantoic fluid of infected chicken embryo and get final product.
3. the influenza A virus strain that preserving number is CGMCCNO.7606 is in preparation H4N6 subtype influenza virus diagnose reagentApplication.
4. the influenza A virus strain that preserving number is CGMCCNO.7606 answering in preparation H4 subtype avian influenza virus vaccineWith.
5. a nucleic acid for separation, is characterized in that, its nucleotide sequence is as shown in SEQIDNO.1.
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| CN101508978A (en) * | 2009-01-04 | 2009-08-19 | 山东省农业科学院畜牧兽医研究所 | Separation identification and purification process for chicken source H9N2 avian influenza virus strain and uses thereof |
| CN101508977A (en) * | 2009-01-04 | 2009-08-19 | 山东省农业科学院畜牧兽医研究所 | Separation identification and purification process for chicken source H9N2 avian influenza virus strain and uses thereof |
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| CN101508978A (en) * | 2009-01-04 | 2009-08-19 | 山东省农业科学院畜牧兽医研究所 | Separation identification and purification process for chicken source H9N2 avian influenza virus strain and uses thereof |
| CN101508977A (en) * | 2009-01-04 | 2009-08-19 | 山东省农业科学院畜牧兽医研究所 | Separation identification and purification process for chicken source H9N2 avian influenza virus strain and uses thereof |
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| 一株野鸭源H4N6亚型禽流感病毒A/mallard/yancheng/2005的全基因组克隆和序列分析;彭宜 等;《畜牧兽医学报》;20091231;第40卷(第4期);548-553 * |
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