CN105039270A - H9N2 subtype bird flu cold adaption attenuated strain and application thereof - Google Patents
H9N2 subtype bird flu cold adaption attenuated strain and application thereof Download PDFInfo
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Abstract
The invention discloses a H9N2 subtype bird flu cold adaption attenuated strain and application thereof. The H9N2 subtype bird flu cold adaption attenuated strain H9AIV-SD/01/10-CA has a preservation number: CGMCC NO. 10908. The H9AIV-SD/01/10-CA strain is attenuated strain, can serve as a live vaccine and has the following advantages compared with current inactivated vaccine: (1) cell-mediated immunity and mucosal immunity with a crossing protection effect can be generated to resist infection of different epidemic strains; (2) cellular immune response can be induced to fast generate a protection effect after immune, and can resist infection of influenza virus in 10-14 days after immune; (3) the inoculation times are less, immune duration is long and the strain is very critical to prevention of H9N2 virus.
Description
Technical field
The present invention relates to a kind of H9N2 subtype avian influenza acclimatization to cold attenuated strain and application thereof.
Background technology
Be separated in the chicken house that Chinese Guangdong is economized first from 1992 and obtain H9N2 influenza virus, this virus is widely current in the poultry and wild bird of China, causes chicken group laying rate and declines and serious polyinfection.Moreover, H9N2 virus also there occurs the case that people infects 1999,2003 and 2013.There are some researches show, the novel H7N9 virus that in March, 2013 is broken out in China causes a large amount of human infections, and cause serious respiratory symptom and higher mortality ratio, and 6 of this H7N9 virus internal gene derive from the popular G57 genotype H9N2 virus of recent advantage.Above-mentioned research shows, the H9N2 virus of poultry of being popular in causes huge financial loss and serious threat to mankind's public health security.
In order to reduce the sickness rate of H9N2 virus in poultry, within 1998, China brings into use the infection of inactivated vaccine control H9N2 virus, uses the initial stage to control the outburst of H9N2 virus and the propagation of virus to a certain extent at vaccine.But H9N2 inactivated vaccine effect declines gradually in recent years.This is because there is a lot of defect in inactivated vaccine.First; inactivated influenza virus vaccine produces antibody for influenza surface protein HA and NA very easily morphed; because influenza antigen variation is fast; usual needs constantly change the strain of inactivated vaccine for resisting the infection of epidemic isolates; the research and development of inactivated vaccine often do not catch up with the speed of virus variation, thus inactivated vaccine often there will be can not the situation of timely and effective control flow check Influenza Virus outburst.The second, inactivated vaccine is difficult to produce mucosa-immune and cellular immunization, and this characteristic makes the protectiveness of vaccine shortage to different epidemic isolates.3rd, after the immunity of commercialization inactivated vaccine, approximately need the protectiveness that could produce infected by influenza for 3 weeks.Generally, chick is initial immunity H9N2 inactivated vaccine when 1 week age, and As time goes on maternal antibody level declines gradually, almost disappears when 3 week age of chicken.Therefore, chick causes to inactivated vaccine and very easily infects H9N2 virus during this period of time before effective immune response after maternal antibody disappears, and inactivated vaccine can not control particularly popular in the broiler chicken H9N2 bird flu of current China aviculture.
Summary of the invention
The object of this invention is to provide a kind of H9N2 subtype influenza virus acclimatization to cold attenuated strain and application thereof.
H9 subtype avian influenza acclimatization to cold virus H9AIV-SD/01/10-CA provided by the invention, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on 06 30th, 2015 and (be called for short CGMCC, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica), preservation registration number is CGMCCNO.10908.H9 subtype avian influenza acclimatization to cold virus H9AIV-SD/01/10-CACGMCCNO.10908, is called for short H9AIV-SD/01/10-CA strain.
The application of H9AIV-SD/01/10-CA strain in the product for the preparation of prevention H9N2 subtype influenza virus also belongs to protection scope of the present invention.
The application of H9AIV-SD/01/10-CA strain in the product of the disease caused for the preparation of prevention H9N2 subtype influenza virus also belongs to protection scope of the present invention.
H9AIV-SD/01/10-CA strain is also belonging to protection scope of the present invention for the preparation of the application in the product of birds flu-preventing.Described bird flu is caused by H9N2 subtype influenza virus.
H9AIV-SD/01/10-CA strain is also belonging to protection scope of the present invention for the preparation of the application in the vaccine for H9N2 subtype influenza virus.
The present invention also protects a kind of for preventing the product of H9N2 subtype influenza virus, and its activeconstituents is H9AIV-SD/01/10-CA strain.
The present invention also protects a kind of product of the disease for preventing H9N2 subtype influenza virus to cause, and its activeconstituents is H9AIV-SD/01/10-CA strain.
The present invention also protects a kind of product for birds flu-preventing, and its activeconstituents is H9AIV-SD/01/10-CA strain.Described bird flu is caused by H9N2 subtype influenza virus.
The present invention also protects a kind of vaccine for H9N2 subtype influenza virus, and its activeconstituents is H9AIV-SD/01/10-CA strain.
Arbitrary described H9N2 subtype influenza virus specifically can be A/chicken/Shandong/sd01/2010, A/chicken/Beijing/3/1999, A/chicken/Hebei/0617/2007, A/chicken/Shandong/ZB/2007, A/chicken/Hebei/YT/2010 or A/chicken/Guangdong/01/2011 above.
Arbitrary described H9N2 subtype influenza virus specifically can be the H9N2 subtype influenza virus of the H9N2 subtype influenza virus of genotype G57, the H9N2 subtype influenza virus of genotype G02, the H9N2 subtype influenza virus of genotype G51, the H9N2 subtype influenza virus of genotype G60 or genotype G68 above.
H9AIV-SD/01/10-CA strain provided by the invention can well copy under 25 DEG C of cold condition, but under 41 DEG C of hot conditionss, grow and be suppressed the normal body temperature of chicken (41 DEG C be), shows good security to the SPF chicken in 3 week age.In addition, challenge test result confirms, this H9AIV-SD/01/10-CA strain can the infection of H9N2 virus to chicken of available protecting different genotype as living vaccine.
Current vaccination is the Main Means of China's prevention and control of fowl influenza epidemic situation.China started to apply the H9N2 influenza virus in inactivated vaccine immunity measure prevention and control chicken group from 1998.But it is slow that inactivated vaccine not only produces antibody speed, and be difficult to inducing mucosal antibody and cellular immunization, be difficult to produce immanoprotection action to antigenic drift strain.H9AIV-SD/01/10-CA strain provided by the invention is attenuated strain, can use as living vaccine, relative to existing inactivated vaccine, there is following advantage: (1) can produce the cellular immunization and mucosal immunity with cross protection effect, resists the infection of different epidemic isolates; (2) can react by inducing cellular immune, produce Vaccine effectiveness rapidly afterwards in immunity, within 10-14 days after immunity, the infection of influenza virus can be resisted; (3) inoculation times is few, and immune duration is long.
The present invention is most important for the control of H9N2 virus.
Accompanying drawing explanation
The result of Fig. 1 acclimatization to cold phenotypic evaluation, * represents the duplicating efficiency of acclimatization to cold virus and wild-type virus, and there were significant differences (P<0.01), and * * * represents the duplicating efficiency of acclimatization to cold virus and wild-type virus, and there were significant differences (P<0.001).
The result of Fig. 2 temperature sensitivity phenotypic evaluation, * * * represents the duplicating efficiency of acclimatization to cold virus and wild-type virus, and there were significant differences (P<0.001).
Embodiment
Following embodiment is convenient to understand the present invention better, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Test materials used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.Quantitative test in following examples, all arranges and repeats experiment for three times, results averaged.
SD/01/10 strain (the representative strain of the preponderant genotype G57 of current H9N2 subtype avian influenza virus, its full name is A/chicken/Shandong/sd01/2010, is labeled as SD/01/10-WT in the present embodiment).
Mention A/chicken/Shandong/sd01/2010, A/chicken/Beijing/3/1999, A/chicken/Hebei/0617/2007, A/chicken/Shandong/ZB/2007, the document of A/chicken/Hebei/YT/2010 and A/chicken/Guangdong/01/2011: Pu, J., Wang, S., Yin, Y., Zhang, G., Carter, R.A., Wang, J., Xu, G., Sun, H., Wang, M., Wen, C., 2014.EvolutionoftheH9N2influenzagenotypethatfacilitatedt hegenesisofthenovelH7N9virus.ProceedingsoftheNationalAca demyofSciences, 201422456..
Mdck cell: China Concord Medical Science University's Institute of Basic Medical Sciences's cell centre.
If no special instructions, the PBS damping fluid in embodiment is the PBS damping fluid of pH7.2,0.1M.
The preparation of embodiment 1, strain and preservation
One, the preparation of strain
By SPF chicken embryo 37 DEG C of hatchings 10 days; Then, with sterile PBS buffer dilution SD/01/10-WT strain to 4 HAU (HAU), 0.2ml/ piece of dose inoculation 3 ~ 5 pieces of chicken embryos, and place 33 DEG C of incubators and hatch 48 hours, discard the dead germ in 24h; Then, place 4 DEG C of refrigerator 12 h before harvest chick embryo allantoic liquids, and do hemagglutination test with the chicken red blood cell of 0.5%, preserve the embryo toxicity that hemagglutinative titer is the highest, and be labeled as 1st generation.
33 DEG C to 25 DEG C continuous passages of progressively lowering the temperature, go down to posterity the virus all using the chick embryo allantoic cavity of 3 ~ 5 piece of 10 age in days to inoculate 4 HAU of sterile PBS buffer dilution at every turn, to hatch after 48 hours at 4 DEG C of refrigerator freezing 12 hr collections allantoic fluids, do hemagglutination test with the chicken red blood cell of 0.5%; Virus goes down to posterity 10 times in each temperature.
After being passaged to 25 DEG C, continuous passage 25 times, finally utilizes limiting dilution assay purified virus again, is about to virus 10 times of doubling dilutions 10
1~ 10
6doubly, each extent of dilution inoculates 3 piece of 10 age in days SPF chicken embryo, 25 DEG C of hatching 48 h before harvest allantoic fluids measure hemagglutinative titer, select blood clotting positive and the chicken embryo results virus that extent of dilution is the highest, and the purifying carried out next time, repeat above-mentioned purification process 9 times, finally obtain in 1 strain acclimatization to cold virus strain, by its called after H9 subtype avian influenza acclimatization to cold virus H9AIV-SD/01/10-CA.
Two, the preservation of strain
H9 subtype avian influenza acclimatization to cold virus H9AIV-SD/01/10-CA, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on 06 30th, 2015 and (be called for short CGMCC, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica), preservation registration number is CGMCCNO.10908.H9 subtype avian influenza acclimatization to cold virus H9AIV-SD/01/10-CACGMCCNO.10908, is called for short H9AIV-SD/01/10-CA strain.
The qualification of embodiment 2, strain
Virus to be measured is H9AIV-SD/01/10-CA strain or SD/01/10-WT strain.
One, viral PFU measuring method
1, in 6 porocyte culture plates, cultivate MDCK cell monolayers, remove the nutrient solution in hole, with PBS buffer solution cell 3 times, discard washing lotion.
2, prepare the EP pipe of 10 1.5mL, often in pipe, add the DMEM substratum that 900 μ L contain 100U/ml penicillin and 100 μ g/ml Streptomycin sulphates; Then, add the virus stock solution used of 100 μ L virus to be measured in the first pipe, fully after mixing, sucking-off 100 μ L to the second manages, and operates the tenth pipe successively, is each dilution viral dilution liquid, carries out mark, be placed in and preserve on ice.
3, get 6 porocyte culture plates of completing steps 1, the viral dilution liquid (every hole 200 μ L) that inoculation step 2 obtains, at 37 DEG C, 5%CO
2quiescent culture 1h in incubator.
4, add TPCK-trypsin in containing in the DMEM substratum of 100U/ml penicillin and 100 μ g/ml Streptomycin sulphates of preheating and mix (TPCK-trypsin concentration is in the medium 3 μ g/mL), then mix with the volume ratio of 4g/100mL aqueous agar solution according to 3:1, obtain covering liquid.
5, after completing steps 3, get described 6 porocyte culture plates, reject culture supernatant, every hole adds the covering liquid of 3ml step 4 preparation, and 4 DEG C of horizontal rest 15-20min, to make it to solidify, then move into CO
2cultivate 24h-72h for 37 DEG C in incubator, then observe plaque test situation.
Observe the method for plaque test situation: each hole adds 1ml formalin, and fixed cell 6 hours, then uses tap water cell hole, glue is thrown away, then every hole adds 300-400 μ l1 ‰ crystal violet solution and leaves standstill 5 minutes, washes out remaining dye liquor with tap water, i.e. visible plaque.
Two, viral EID
50measuring method
1,9-11 age in days SPF chicken embryo is placed in darkroom, observes with candler, mark the air chamber of each chicken embryo, avoid great vessels and mark.
2, prepare 10 1.5mLEP pipes, often in pipe, add the PBS damping fluid that 900 μ L contain 100U/ml penicillin and 100 μ g/ml Streptomycin sulphates; Then, add the virus stock solution used of 100 μ L virus to be measured in the first pipe, fully after mixing, sucking-off 100 μ L to the second manages, and operates the tenth pipe successively, is each dilution viral dilution liquid, carries out mark, be placed in and preserve on ice.
3, by the chicken embryo marked with 75% alcohol carry out disinfection, punch with part place above air chamber with the punch tool after sterilization, inject the viral dilution liquid (each dilution viral dilution liquid inoculates three chicken embryos) that 0.1mL step 2 obtains, paraffin sealing is placed on incubator and hatches, per sunshine egg.
4, after virus inoculation diluent 48-72h, chicken embryo is placed in 4 DEG C of refrigerator 1h, then uses 75% alcohol disinfecting air chamber, open with tweezers, draw allantoic fluid to the 96 hole blood-coagulation-board of 25 μ L, carry out hemagglutination test, and record result.
By Reed-Muench method formula, calculate median infective dose, the correction factor of 10 times of serial dilutions is 1.
Log
10eID
50=higher than the viral dilution denary logarithm+respective distance ratio × dilution factor denary logarithm of 50% infection rate.
Three, acclimatization to cold phenotypic evaluation
1, by the SPF chicken embryo (0.2mL/ piece) of virus inoculation to 10 age in days to be measured for 4HAU, then chicken embryo 25 DEG C is hatched 48 hours, collect allantoic fluid, detect virus titer (EID
50/ mL); The re-treatment of 5 pieces of SPF chicken embryos is set.
2, by the SPF chicken embryo (0.2mL/ piece) of virus inoculation to 10 age in days to be measured for 4HAU, then chicken embryo 35 DEG C is hatched 48 hours, collect allantoic fluid, detect virus titer (EID
50/ mL); The re-treatment of 5 pieces of SPF chicken embryos is set.
The standard of perfection of acclimatization to cold phenotype is: virus titer≤100 that virus titer/step 1 that step 2 obtains obtains.
(experimental data is the mean value ± SD of three independent experiments to the results are shown in Figure 1; *, P<0.01; * *, P<0.001).
Under 25 DEG C and 35 DEG C of culture condition, H9AIV-SD/01/10-CA strain is equal well-grown in chicken embryo, and virus titer reaches 10 respectively
6.25eID
50/ ml and 10
7.25eID
50/ ml.Under 25 DEG C of culture condition, SD/01/10-WT strain cannot grow in chicken embryo.Result shows, H9AIV-SD/01/10-CA strain obtains acclimatization to cold characteristic.
Four, temperature sensitivity phenotypic evaluation
1, prepare the EP pipe of 10 1.5mL, often in pipe, add the DMEM substratum that 900 μ L contain 100U/ml penicillin and 100 μ g/ml Streptomycin sulphates; Then, add the virus stock solution used of 100 μ L virus to be measured in the first pipe, fully after mixing, sucking-off 100 μ L to the second manages, and operates the tenth pipe successively, is each dilution viral dilution liquid, carries out mark, be placed in and preserve on ice.
2, add TPCK-trypsin in containing in the DMEM substratum of 100U/ml penicillin and 100 μ g/ml Streptomycin sulphates of preheating and mix (TPCK-trypsin concentration is in the medium 3 μ g/mL), then mix with the volume ratio of 4g/100mL aqueous agar solution according to 3:1, obtain covering liquid; Mdck cell is seeded to 6 orifice plates, the viral dilution liquid inductance dye MDCK cell monolayers (37 DEG C adsorb 1 hour) then step 1 obtained, then discard culture supernatant, every hole adds 3ml covering liquid, and 4 DEG C of horizontal rest 15-20min make it to solidify.
3, after completing steps 2, get described 6 orifice plates, be placed in 33 DEG C, 5%CO
2incubator hatches 48 hours, covering liquid is shifted out, and detects virus titer (PFU).
4, after completing steps 2, get described 6 orifice plates, be placed in 37 DEG C, 5%CO
2incubator hatches 48 hours, covering liquid is shifted out, and detects virus titer (PFU).
5, after completing steps 2, get described 6 orifice plates, be placed in 41 DEG C, 5%CO
2incubator hatches 48 hours, covering liquid is shifted out, and detects virus titer (PFU).
The standard of perfection of temperature sensitivity: the virus titer > 100 obtained in virus titer/step 5 that step 3 obtains.
(experimental data is the mean value ± SD of three independent experiments to the results are shown in Figure 2.***,P<0.001)。At 33 DEG C, the ability that H9AIV-SD/01/10-CA strain and SD/01/10-WT strain form plaque does not have difference.At 37 DEG C, the ability that H9AIV-SD/01/10-CA strain forms plaque is obviously weaker than SD/01/10-WT strain.At 41 DEG C, H9AIV-SD/01/10-CA strain can not form plaque.H9AIV-SD/01/10-CA strain can effectively copy 33 DEG C and 37 DEG C, but does not copy under 41 DEG C of conditions.SD/01/10-WT strain all can effectively copy at 33 DEG C, 37 DEG C and 41 DEG C, and virus titer is similar.The above results shows: H9AIV-SD/01/10-CA strain has possessed the temperature sensitivity feature of acclimatization to cold attenuated live vaccine.
Embodiment 3, strain are as the immunogenicity of living vaccine and protectiveness evaluation
H9N2 subtype avian influenza virus is attacked strain and is respectively: A/chicken/Shandong/sd01/2010, A/chicken/Beijing/3/1999 (the representative strain of genotype G02), A/chicken/Hebei/0617/2007 (the representative strain of genotype G51), A/chicken/Shandong/ZB/2007 (the representative strain of genotype G60), A/chicken/Hebei/YT/2010 (the representative strain of genotype G57) and A/chicken/Guangdong/01/2011 (the representative strain of genotype G68).
Experimental subjects is the SPF Leghorn (being purchased from Beijing Cimmeria Wei Tong experimental technique company limited) in 1 week age.
Attack strain for often kind to test as follows respectively:
Immune group: get 3 experimental subjectss, (Nasal immunization, immunizing dose is 10 to single immunization H9AIV-SD/01/10-CA strain
6eID
50, immune volume is 200 μ l), these 3 experimental subjectss belong to direct infection group; Get 3 experimental subjectss, single immunization H9AIV-SD/01/10-CA strain Nasal immunization, immunizing dose is 10
6eID
50, immune volume is 200 μ l), these 3 experimental subjectss belong to propagation group of living together; It is 10 that direct infection group experimental subjects immunity H9AIV-SD/01/10-CA strain inoculated 0.2ml concentration by the mode of collunarium eye droppings after 2 weeks
6eID
50the H9N2 subtype avian influenza virus of/ml attacks strain virus liquid; Inoculation is made to attack the direct infection group experimental subjects of strain after 24 hours and the live together propagation group experimental subjects of immune H9AIV-SD/01/10-CA strain after 2 weeks is lived together; Attack strain after 3 days, after 5 days and within 7 days, gather buccal swab and the cloacal swab of each experimental subjects afterwards respectively at inoculation, detect the virus titer (EID in swab
50/ mL).
Control group first: get 3 experimental subjectss, the isopyknic PBS damping fluid of single immunization, these 3 experimental subjectss belong to direct infection group; Get 3 experimental subjectss, the isopyknic PBS damping fluid of single immunization, these 3 experimental subjectss belong to propagation group of living together; It is 10 that direct infection group experimental subjects immunity PBS damping fluid inoculated 0.2ml concentration by the mode of collunarium eye droppings after 2 weeks
6eID
50the H9N2 subtype avian influenza virus of/ml attacks strain virus liquid; Inoculation is made to attack the direct infection group experimental subjects of strain after 24 hours and the live together propagation group experimental subjects of PBS damping fluid after 2 weeks is lived together; Attack strain after 3 days, after 5 days and within 7 days, gather buccal swab and the cloacal swab of each experimental subjects afterwards respectively at inoculation, detect the virus titer (EID in swab
50/ mL).
Control group second: get 3 experimental subjectss, and SD/01/10-WT strain after single immunization deactivation (Nasal immunization, immune volume is 200 μ l; Before deactivation, the viral level of every 200 μ l virus liquids is 10
6eID
50; Ablation method for add 0.1 volume formaldehyde in every 100 volume virus liquids, then 37 DEG C of standing 16-18 hour), these 3 experimental subjectss belong to direct infection group; Get 3 experimental subjectss, the SD/01/10-WT strain after single immunization deactivation, these 3 experimental subjectss belong to propagation group of living together; It is 10 that SD/01/10-WT strain after direct infection group experimental subjects immunological sterilization inoculated 0.2ml concentration by the mode of collunarium eye droppings after 2 weeks
6eID
50the H9N2 subtype avian influenza virus of/ml attacks strain virus liquid; Inoculation is made to attack the direct infection group experimental subjects of strain after 24 hours and the live together propagation group experimental subjects of the SD/01/10-WT strain after immunological sterilization after 2 weeks is lived together; Attack strain after 3 days, after 5 days and within 7 days, gather buccal swab and the cloacal swab of each experimental subjects afterwards respectively at inoculation, detect the virus titer (EID in swab
50/ mL).
The results are shown in Table 1.
To the protective efficacy that different H9N2 subtype avian influenza virus infects after the immunity of table 1 vaccine strain
The effective immune protection effectiveness of vaccine to the influenza virus of different genotype is the important component part of vaccine evaluation.For this reason, contriver evaluates its immune efficacy by immune chicken protest test.Except the immune chicken of A/chicken/Shandong/ZB/2007 infected group detects lower virus titer in the oral cavity of 3 days after virus infection, other immune chickens all do not have toxin expelling after virus infection.Result confirms, strain of the present invention (after single immunization 2 weeks) can produce the resistibility for different genotype H9N2 virus infection rapidly as after living vaccine immunity, there is the feature of security height and good immune effect, for the control of H9N2 influenza virus is laid a good foundation.
Embodiment 4, strain are as the safety evaluation of living vaccine
1, security
With the mode of collunarium eye droppings SD/01/10-WT strain, H9AIV-SD/01/10-CA strain inoculated respectively 10 3 week age SPF Leghorn, dosage of inoculation is 10
6.6eID
50(0.2ml/ only.Establish negative control group in addition, 0.2mlPBS damping fluid is given respectively 10 SPF Leghorn collunarium eye droppings inoculations in 3 week age.Observe the clinical symptom infecting all chickens in latter 14 days.
The chicken of inoculation SD/01/10-WT shows spiritual depressed after inoculation in 2-7 days, appetite declines, and eye nasal discharge increases, the symptom of diarrhea.The mental status, appetite are all normal after inoculation for the chicken of inoculation H9AIV-SD/01/10-CA.
2, the detection of virus replication in SPF chicken body
In the mode of collunarium eye droppings, SPF Leghorn in 3 week age is inoculated SD/01/10-WT, H9AIV-SD/01/10-CA respectively, dosage of inoculation is 10
6eID
50(0.2ml/ only).Within 2,3,5,7 days, put to death 6 after inoculation respectively, get lungs and tracheae.Lungs and tracheae add containing 100U/ml penicillin and 100 μ g/ml Streptomycin sulphates sterile PBS buffer grind, and use EID
50method carry out quantitatively organizing virus titer contained by internal organs.
The results are shown in Table 2.
The replication of table 2 strain in 3 week age SPF chicken
Internal organs number/internal organs sum (the log of virus detected
10eID
50the mean+SD of/ml)
Note:
binfect size of animal/this test group of animals total quantity of virus;
cvirus titer is expressed as average titer ± difference value (log
10eID
50/ ml).
3, the detection of virus transmission capacity in SPF chicken group
In the mode of collunarium eye droppings, SPF Leghorn in 3 week age is inoculated SD/01/10-WT, H9AIV-SD/01/10-CA respectively, dosage of inoculation is 10
6eID
50(0.2ml/ only), as direct infection group.SPF Leghorn in 3 of 6 same batch week age is put into shield retaining and is carried out raising of living together, as propagation group of living together by the rear 24h of inoculation.2 days and 4 days gather throat swabs and cloacal swab after inoculation, with containing 100U/ml penicillin and 100 μ g/ml Streptomycin sulphates sterile PBS buffer dissolve, and use EID
50method carry out quantitatively to virus titer contained in swab, lowest detection titre is 10
-1eID
50/ mL.
The results are shown in Table 3.
The transmission capacity of table 3 virus in 3 week age SPF chicken group
Note:
bswab number/this experimental group isolating virus gathers swab number;
cvirus titer is expressed as average titer ± difference value (log
10eID
50/ ml).
The prerequisite that attenuated live vaccine can be applied is the security that must ensure vaccine.Research finds, H9AIV-SD/01/10-CA only shows transient infection after inoculating the SPF chicken in 3 week age, virus replication is only confined to the upper respiratory tract, in addition the ability do not propagated between chicken group of this virus, shows that H9AIV-SD/01/10-CA is safe as living vaccine to chicken.
Claims (10)
1.H9 subtype avian influenza acclimatization to cold virus H9AIV-SD/01/10-CA, its deposit number is CGMCCNO.10908.
2. the application of virus described in claim 1 in the product for the preparation of prevention H9N2 subtype influenza virus.
3. the application of virus described in claim 1 in the product of the disease caused for the preparation of prevention H9N2 subtype influenza virus.
4. virus described in claim 1 is for the preparation of the application in the product of birds flu-preventing.
5. virus described in claim 1 is for the preparation of the application in the vaccine for H9N2 subtype influenza virus.
6., for preventing a product for H9N2 subtype influenza virus, its activeconstituents is virus described in claim 1.
7., for a product for the disease of preventing H9N2 subtype influenza virus to cause, its activeconstituents is virus described in claim 1.
8., for a product for birds flu-preventing, its activeconstituents is virus described in claim 1.
9., for a vaccine for H9N2 subtype influenza virus, its activeconstituents is virus described in claim 1.
10. application as claimed in claim 4 or product as claimed in claim 8, is characterized in that: described bird flu is caused by H9N2 subtype influenza virus.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108300702A (en) * | 2018-02-05 | 2018-07-20 | 山东省农业科学院畜牧兽医研究所 | One plant of chicken source H 9 N 2 avian influenza virus acclimatization to cold strain screening technique and its application |
| CN114836390A (en) * | 2022-06-13 | 2022-08-02 | 华南农业大学 | H9N2 subtype avian influenza virus MDCK cell cold-adaptation attenuated live vaccine strain and cultivation method and application thereof |
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| CN103740654A (en) * | 2014-01-27 | 2014-04-23 | 中国医学科学院医学生物学研究所 | Cold adaptation strain of influenza B virus Vero cell and application thereof |
| CN103898066A (en) * | 2014-01-27 | 2014-07-02 | 中国医学科学院医学生物学研究所 | Vero (Rabies Purified Vaccine for Human Use) cell cold-adapted strain of influenza A virus and application thereof |
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| CN103740654A (en) * | 2014-01-27 | 2014-04-23 | 中国医学科学院医学生物学研究所 | Cold adaptation strain of influenza B virus Vero cell and application thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108300702A (en) * | 2018-02-05 | 2018-07-20 | 山东省农业科学院畜牧兽医研究所 | One plant of chicken source H 9 N 2 avian influenza virus acclimatization to cold strain screening technique and its application |
| CN108300702B (en) * | 2018-02-05 | 2021-09-14 | 山东省农业科学院畜牧兽医研究所 | Chicken-derived H9N2 avian influenza virus cold-adapted strain screening method and application thereof |
| CN114836390A (en) * | 2022-06-13 | 2022-08-02 | 华南农业大学 | H9N2 subtype avian influenza virus MDCK cell cold-adaptation attenuated live vaccine strain and cultivation method and application thereof |
| CN114836390B (en) * | 2022-06-13 | 2023-05-02 | 华南农业大学 | H9N2 subtype avian influenza virus MDCK cell cold-adaptation attenuated live vaccine strain and application thereof |
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