CN105441393A - Infectious bursal disease very virulent DF-1 cell adapted strain and application thereof - Google Patents
Infectious bursal disease very virulent DF-1 cell adapted strain and application thereof Download PDFInfo
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Abstract
The invention provides an infectious bursal disease very virulent DF-1 cell adapted strain and the application thereof. The infectious bursal disease very virulent DF-1 cell adapted strain is named IBDV-LY23 strain, and the preservation serial number is CGMCC No.11594. The strain can be cultured in a continuous passage mode on DF-1 cells, TCID50 reaches 108.5/mL, vaccines can be prepared after the strain is inactivated, detection proves that immunized chickens have high immunogenicity, 100% protection rate is realized for fatal attack of very virulent viruses IBD/SD/LY/2014, and meanwhile, safety performance is high, efficacy is stable and durability is high. The strain IBDV-LY23 can serve as a vaccine strain to prevent the infectious bursal disease and has great value for preventing and controlling prevalence of the infectious bursal disease very virulent virus.
Description
Technical field
The present invention relates to animal virology field, specifically, relate to Very virulent infectious bursal disease virus DF-1 cell adapted strain and application thereof.
Background technology
Infectious bursal disease (InfectiousBursalDisease, IBD) is a kind of high degree in contact sexually transmitted disease of the chicken caused by bursal disease virus (IBDV).Bursa of Fabricius virus belongs to birnavirus section, Avibirnavirus.Bursal disease virus has two serotypes, and I type bursal disease virus has virulence to chicken, and II C-type virus C is to chicken had no pathogenicity.This disease was imported China in 1979 and starts pandemic, along with the application of bursa of fabricius vaccine, this disease obtains control to a certain degree, but show according to epidemiology survey result, this disease presented the epidemic characteristic made new advances in the last few years: morbidity age in days obviously broadens, have the report of 3 age in days morbidities the earliest, the longest laying hen extending to 200 ages in days, and high age in days chicken group suffers from the report that infectious bursal disease has the regional outburst of formation; Sickness rate and case fatality rate all increase, and morbidity chicken group mostly is vaccinated flock, and this feature illustrates, I C-type virus C exists variant and highly virulent strain (vvIBDV), can break through the protection of traditional vaccine.In chicken blood, the antibody of traditional vaccine plant height titre can not resist the attack of vvIBDV completely.Clinical symptom is identical with classical strains, and pathology is more serious, can make the fabricius bursa, thymus gland, spleen and marrow major injury; The infectivity of IBDV strengthens to some extent, route of transmission is increased, IBDV prolonged survival period in vivo and in external environment, and the resistivity of various physic-chemical changes strengthens to some extent to external world, the route of transmission of this disease is caused to be increased, by digestive tube and respiratory tract and other various forms contact infections; Secondary or accompanying infection are serious, because the fabricius bursa is central immune organ, can cause body immunosuppression in various degree after therefore infecting infectious bursal disease, chicken group are grown produce being obstructed in various degree; In addition because environmental health is poor, feeding and management is chaotic, and feed nutritive value is incomplete, can cause the serious secondary or concurrent such as intestinal bacteria, mycoplasma, coccidiosis, add difficulty, mortality ratio is increased to Diagnosis and Treat.For this situation; be separated up-to-date fabricius bursa virulent epidemic isolates; screening, cultivate immunogenicity good and can adapt to cell line proliferates kind poison; be applied to large-scale production, the IBD vaccine product of development and production actual needs is extremely important for the prevention and corntrol of this disease.
Summary of the invention
The object of this invention is to provide Very virulent infectious bursal disease virus DF-1 cell adapted strain (i.e. chicken infectivity bursa of Fabricius virus IBDV-LY23 strain) and application thereof.
In order to realize the object of the invention, there is sick chicken bursa, the spleen tissue of classical symptom and pathological change in the chicken house collection that doubtful IBD epidemic situation occurs from Shandong Linyi Prefecture the present inventor, after standardized method process, is separated and obtains CK/SD/LY/2014 strain.Pathogenicity is carried out to this strain isolated, has cloned its main protection antigen gene VP2, and carried out sequential analysis and compared.Result shows, this strain isolated VP2 gene has the highly virulent strain amino acids characteristic such as 222A, 242I, 279D, 256I, 284A, 294I and 299S, be 94.9%, 95.1% with vaccine strain B87, D78 nucleotide acid homology, be 97.6% with European standard virulent UK661 strain nucleotide homology, amino acid identity is 99.6%.It is to SPF chicken embryo medium lethal dose (ELD
50) be 10
7.17/ 0.2ml, with 10
4eLD
50/ dose inoculation SPF chicken in 4 week age only, mortality ratio reaches 90%.Thus confirm that infectious bursal disease virus CK/SD/LY/2014 is the domestic highly virulent strain that a strain meets European virulent standard and feature.
This CK/SD/LY/2014 strain is after chicken embryo blind passage 5 generation, and again through chick fibroblast DF-1 continuous passage 15 generation, result shows that virus is stably bred in a large number on cell, the ELD of virus to 10 age in days SPF chicken embryos
50be 10
6.38/ 0.2ml, cell median infective dose (TCID
50) be 10
8.5tCID
50/ ml.And then screening obtains Very virulent infectious bursal disease virus DF-1 cell adapted strain of the present invention, called after IBDV-LY23 strain, Classification And Nomenclature is: chicken infectivity bursa of Fabricius virus, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, address, Institute of Microorganism, Academia Sinica, postcode 100101, deposit number CGMCCNo.11594, preservation date on November 19th, 2015.
Very virulent infectious bursal disease virus DF-1 cell adapted strain IBDV-LY23 of the present invention is carried out Batch Culture in the mode synchronously connecing poison; results virus; vaccine is prepared into by after its deactivation; through detecting, there is good immunogenicity after immunity chicken body; to the lethal hit of virulent IBD/SD/LY/2014, there is the protection ratio of 100%.In the comparative studies that vaccine similar with domestic and international part carries out, this virulent deactivation vaccine antibody positive rate, antibody titers and the protection ratio to virulent, participating in the vaccine compared, all occuping the first.
The present invention also provides described Very virulent infectious bursal disease virus DF-1 cell adapted strain preparing the application in bursal disease vaccine.
Aforesaid application, mixes the inactivation of viruses liquid of IBDV-LY23 strain by a certain percentage with adjuvant and obtains bursal disease vaccine.
Wherein, beta-propiolactone deactivation is preferably used in described IBDV-LY23 strain.Described adjuvant is MONTANIDEISA71VG.
Further, aforementioned applications is specially: by the deactivation of IBDV-LY23 strain beta-propiolactone, mixed by inactivation of viruses liquid, obtain described vaccine through emulsification with MONTANIDEISA71VG according to weight ratio 3:7.
The present invention also provides a kind of vaccine composition preventing infectious bursal disease, and the effective constituent of described vaccine composition comprises the Very virulent infectious bursal disease virus DF-1 cell adapted strain after deactivation.
Very virulent infectious bursal disease virus DF-1 cell adapted strain IBDV-LY23 provided by the invention can cultivate on DF-1 cell in continuous passage, TCID
50reach 10
8.5/ mL, is prepared into vaccine by after its deactivation, has good immunogenicity, to the lethal hit of virulent IBD/SD/LY/2014, has the protection ratio of 100%, security is simultaneously high, effect is stablized, the extended period is long after immune chicken body through detecting.Strain IBDV-LY23 of the present invention can be prevented infectious bursal disease as vaccine strain, to prevention, control that infections chicken cloacal bursa virulent is popular has important value.
Accompanying drawing explanation
Fig. 1 is the RT-PCR detected result of CK/SD/LY/2014 strain VP2 gene in the embodiment of the present invention 2; Wherein, the RT-PCR product of M to be Trans2KplusMarkerS:1 be VP2 gene.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.If do not specialize, the conventional means that technique means used in embodiment is well known to those skilled in the art, is raw materials usedly commercial goods.
Embodiment 1 infectious bursa of Fabricius virus CK/SD/LY/2014 strain Isolation and ldentification
In August, 2014, there is doubtful IBD epidemic situation in Shandong Linyi Prefecture chicken house, there is sick chicken bursa, the spleen tissue of classical symptom and pathological change in aseptic collection, shred grinding, weight ratio respectively according to 1:3 after weighing adds the PBS of sterilizing, 4 DEG C, the centrifugal 10min of 6000r/min, 4 DEG C, the centrifugal 10min of 8000r/min, getting supernatant liquor proceeds in another aseptic 1.5ml centrifuge tube, add dual anti-(final concentration is penicillin 2000IU/ml, Streptomycin sulphate 2mg/ml), 37 DEG C of effect 1h, each 5 pieces of 10 age in days SPF chicken embryo is inoculated, 0.2mL/ piece through chorioallantoic membrane.Discard chicken embryo dead in 24h, observe 1 time every 6h later, collect dead chicken embryo, take out all chick chorioallantoic membranes and idiosome to 144 hours, tissue mashing, do 2 times of dilutions with the aseptic PBS containing mycillin, be designated as E1 generation, keep sample detection, and viral isolates is placed in-70 DEG C of preservations.
Agar diffusion test (AGP):
Above-mentioned E1 is carried out agar diffusion test for viral centrifuging and taking supernatant liquor and IBD standard positive serum, and establishes standard antigen and standard positive serum control wells, observations place 24h in 37 DEG C of incubators after.About for having there is the clear precipitation line of oyster white in 36h, E1 between vial supernatant and standard positive serum hole, identical with positive controls, and negative control group is without precipitation, and result shows that IBDV is positive.By its called after CK/SD/LY/2014 strain.
Embodiment 2 infectious bursa of Fabricius virus CK/SD/LY/2014 strain Biological characteristics
Egg inoculation is tested:
With chorioallantoic membrane (CAM) inoculation method, above-mentioned E1 is inoculated in 10 piece of 9 ~ 10 age in days SPF chicken embryo for vial supernatant by every embryo 0.2mL, if isodose aseptic PBS liquid is negative control group.Put 37 DEG C of incubators and continue hatching, every 12h checks 1 time according to egg, discards dead embryo in 24h, record chicken embryo death and pathology situation, and inoculation is observed to 144h, collects all survival chicken embryos and observes pathology.Chicken embryo after inoculation 48 ~ 72h is interior dead, and dead embryo chorioallantoic membrane thickens, belly swelling, cuts open inspection visible, the yellow sex change of chicken embryo liver, and downright bad with mottling, conforms to pathology caused by IBDV.
RT-PCR detects:
Utilize nucleic acid extraction test kit to extract IBDV nucleic acid, carry out RT-PCR according to Promega company AMV ThermoScript II product description and detect and VP2 gene amplification.Through RT-PCR amplification, after electrophoresis, there is the band of 1 treaty 1300bp size, meet expection size (Fig. 1).Isolated strain VP2 gene nucleotide series and corresponding aminoacid sequence are analyzed, result shows that this strain isolated VP2 gene has the highly virulent strain amino acids characteristic such as 222A, 242I, 279D, 256I, 284A, 294I and 299S, be 94.9%, 95.1% with vaccine strain B87, D78 nucleotide acid homology, be 97.6% with European standard virulent UK661 strain nucleotide homology, amino acid identity is 99.6%.
CK/SD/LY/2014 strain VP2 the 212nd amino acids is undergone mutation (D → N), and the variation (D → N) of 212 amino acids of IBDV epidemic strain on VP2 is distinguishing features of the new strain isolated of recent year IBDV.
Pathogenic Tests:
Chicken embryo medium lethal dose (ELD
50) mensuration:
By E1 for vial supernatant, inoculate 10 age in days SPF chicken embryos through chorioallantoic membrane (CAM) approach, by 10 times of dilutions of going forward one by one, get 10
-4~ 10
-10totally 7 extent of dilution, each extent of dilution inoculates 5 pieces of chicken embryos, and dosage of inoculation is 0.2mL/ piece, continues hatching at 37 DEG C of incubators, discards chicken embryo dead in 24h.Per sunshine, embryo 12h shone egg 1 time later, observed to 168h, recorded each group of chicken embryo death situation and idiosome pathology in detail, calculated the ELD of virus in chorioallantoic membrane suspension according to Reed-Muech
50be 10
7.17/ 0.2mL (table 1).
Table 1CK/SD/LY/2014 strain virus titer determination
Chicken body Orthogonal Rotational Regressive Tests:
By 30 4 week age SPF chicken be divided into two groups at random, one group 20, per oral inoculation E1 for vial supernatant, every 0.2mL (10
4eLD
50), another group 10, the sterilizing PBS of identical approach inoculation equivalent is as Normal group.After inoculation, every day observes each group of chicken clinical manifestation, and to dying of illness, chicken cuts open inspection, and records dead quantity and pathological change.
After inoculation, 2d chicken starts morbidity, and chicken group sickness rate reaches 100%, and sick chicken feather is fluffy, and minimizing of searching for food, flocks together, One's spirits are drooping.After inoculation, 3d starts dead successively, and its peak mortality 3d ~ 4d after inoculation, mortality ratio reaches 91.67%.Cut open the visible fabricius bursa swelling of inspection, inside have caseous substance or clot, most fabricius bursa is in " purple grape shape ", and leg, chest muscle have hemorrhage, kidney swelling and have urate deposition.Control group all normal (table 2).
Table 2CK/SD/LY/2014 strain isolated is to the pathogenicity of SPF chicken in 4 week age
The screening of embodiment 3 cell adapted strain and qualification
By E1 for CK/SD/LY/2014 virus liquid with chicken embryo blind passage to E5 generation.By DF-1 cell according to 3 × 10
5the dosage in/hole is laid on 6 orifice plates, and E5 is carried out 10 times of gradient dilutions to 10 for virus liquid DMEM
-4~ 10
-8.The DF-1 cell growing to individual layer is rinsed 2 times with the DMEM not containing serum, and then the virus liquid diluted is inoculated in DF-1 cell, each extent of dilution inoculates 3 holes, 0.3mL/ hole, 37 DEG C of absorption 1.5h, tipping residue virus liquid.Overlay the first layer nutrient agar medium that about 3mm is thick afterwards, put CO
2incubator, 37 DEG C, 5%CO
2under condition, after inversion cultivation 48h, (when pathology appears in cell) overlays the nutrient agar medium of the second layer containing 0.01% toluylene red, 37 DEG C, 5%CO
2under condition, lucifuge is inverted and is cultivated.After Plaque Formation, use water dropper sucking-off, put into the serum-free DMEM of 0.5mL together with agarose, multigelation 3 times, make virus fully release.So repeatedly by virus strain after 3 purifying, picking 5 plaques, being inoculated in individual layer DF-1 cell respectively, to carry out virus expansion numerous.Measure the viral level expanding numerous virus, select to plant malicious F1 generation based on the highest plaque poison of viral level, reach F15 generation continuously by DF-1 cell, called after IBDV-LY23.
Steriling test:
Detected for virus liquid sulphur glycollate culture medium (TG) and peptone from casein agar (GA) (according to " Chinese veterinary pharmacopoeia " annex method) by F15, result display, without colony growth, illustrates in described virus liquid not containing bacterium.
Detected for virus liquid dextrose peptone medium (GP) (according to " Chinese veterinary pharmacopoeia " annex method) by F15, result display, without colony growth, illustrates in described virus liquid not containing mould and saprophytic microorganism.
Specific assay:
The F15 of results is diluted to 10 for the aseptic PBS of virus liquid
4eLD
50/ 0.1ml, mixes with the resisting chicken bursa specific serum of equivalent, and in 37 DEG C and after 60 minutes, six orifice plates of individual layer DF-1 cell are covered with in inoculation, 0.2mL/ hole, and setting simultaneously connects malicious control group 10
4eLD
50/ 0.1ml, 0.1mL/ hole, aseptic PBS, the 0.1mL/ hole of negative control group.Inoculate rear 37 DEG C cultivate 168 hours, every day observation of cell pathology situation.Pathology that neutralization group, negative control group are acellular, connects the porose typical cytopathic that all occurs of poison group, illustrates that this strain is chicken infectivity bursa of Fabricius virus, have specificity, called after chicken infectivity bursa of Fabricius virus IBDV-LY23 strain.
Chicken embryo medium lethal dose (ELD
50) mensuration:
By F15 for vial supernatant, inoculate 10 age in days SPF chicken embryos through chorioallantoic membrane (CAM) approach, by 10 times of dilutions of going forward one by one, get 10
-4~ 10
-10totally 7 extent of dilution, each extent of dilution inoculates 5 pieces of chicken embryos, chooses every embryo 0.2mL, continues hatching at 37 DEG C of incubators, discards chicken embryo dead in 24h.Per sunshine, embryo 12h shone egg 1 time later, observed to 168h, recorded each group of chicken embryo death situation and idiosome pathology in detail, finally calculated the ELD of virus in every 0.2mL chorioallantoic membrane suspension according to Reed-Muech
50.According to chicken embryo death situation, Reed-Muech method is utilized to calculate the ELD of virus in every 0.2mL chorioallantoic membrane suspension
50reach 10
6.38(table 3).
Table 3 chicken embryo medium lethal dose (ELD
50) measure
IBDV-LY23 strain cell median infective dose (TCID
50) measure:
By F15 for virus liquid doubling dilution, get 10
-5, 10
-6, 10
-7, 10
-8, 10
-9totally 5 extent of dilution, inoculate the individual layer DF-1 cell on 96 porocyte plates respectively, inoculation by most high dilution, each extent of dilution inoculates 8 holes, put 37 DEG C hatch 144 hours after, with inoculating cell, hole-specifically observation of cell pathology, occurs that typical cytopathic is judged to infection, calculate viral level according to Reed-Muench method.It is 10 that result display F15 generation plants malicious viral level
8.5tCID
50/ mL.
Exogenous virus detects:
The F15 of results is diluted to 10 for the aseptic PBS of virus liquid
4eLD
50/ 0.1ml, mixes with the resisting chicken bursa specific serum of equivalent, in 37 DEG C and after 60 minutes.Get 10 age in days SPF chicken 20, inoculate with the approach of collunarium, eye droppings, every chicken inoculation 0.2mL, after 21 days, as stated above with dosage repeated inoculation 1 time.1st inoculation latter 42 days blood sampling separation of serum, blood clotting is used to suppress (HI) respectively, fine jade expands (AGP), enzyme linked immunosorbent assay (ELISA), neutralization test (VN), the methods such as immunofluorescence (IFA), detect chicken infectivity bursa of Fabricius virus (IBDV) in serum, Marek's disease poison (MDV), newcastle disease virus (NDV), REV (reticuloendotheliosivirus virus) (REV), avian infectious bronchitis virus (IBV), avian influenza virus (AIV), avian leukosis virus (ILV), avian infectious laryngotracheitis virus (LTV), avian encephalomyclitis virus (AEV), the antibody positive rate of bird pox virus (POX).In serum except infectious bursa of Fabricius virus antibody being detected, without other antiviral antibody, prove that kind of poison is pure, without the pollution of other exogenous virus.SPF chicken is used to test, reliable results.
Table 4 exogenous virus serology results
Toxicity test to SPF chicken:
By 15 4 week age SPF chicken be divided into two groups at random, one group 10, per oral inoculation E1 for vial supernatant, every 0.2mL (10
4eID
50), another group 5, the sterilizing PBS of identical approach inoculation equivalent is as Normal group.After inoculation, every day observes each group of chicken clinical manifestation, and to dying of illness, chicken cuts open inspection, and records dead quantity and pathological change.
After inoculation, 2d chicken starts morbidity, and chicken group sickness rate reaches 100%, and sick chicken feather is fluffy, and minimizing of searching for food, flocks together, One's spirits are drooping.After inoculation, 3d starts dead successively, and its peak mortality 3d ~ 4d after inoculation, mortality ratio reaches 90.00%.Cut open the visible fabricius bursa swelling of inspection, inside have caseous substance or clot, most fabricius bursa is in " purple grape shape ", and leg, chest muscle have hemorrhage, kidney swelling and have urate deposition.Control group all normal (table 5).
Table 5CK/SD/LY/2014 strain isolated is to the pathogenicity of SPF chicken in 4 week age
The preparation of embodiment 4 inactivated vaccine
Virus liquid is tired TCID
50>=10
5/ mL can be used for preparing vaccine.Through the beta-propiolactone 4 DEG C effect 24h that final concentration is 1:2000,37 DEG C of effect 2h, obtain inactivation of viruses liquid.Be that 3:7 mix with MONTANIDEISA71VG according to weight ratio by inactivation of viruses liquid, obtain IBDV-LY23 strain vaccine through emulsification.Vaccine steriling test is undertaken by 2010 editions " Chinese veterinary pharmacopoeias ".
Safety verification: 21 age in days SPF chickens 20,10 each neck dorsal sc injection vaccine 1mL, another 10 compare, and the same terms is raised, and observes and carries out cuing open inspection after 15 days.Vaccine inoculation site tissue, the fabricius bursa and each internal organs are all without observing pathological change.
Minimum immune dosage measures:
Only inoculate each 10 of 21 age in days SPF chicken with 0.1mL, 0.2mL, 0.3mL, 0.4mL, 0.5mL/ respectively with IBDV-LY23 strain vaccine, another 5 inoculation sterilizing PBS are as negative control.Within 7,14,21,28 days, gather serum respectively after immunity, detect antibody.Immune latter 28 days with 10
4eID
50virulent IBD/SD/LY/2014 carries out attacking poison, to attack malicious protection ratio determination minimum immune dosage.Within 7 ~ 14 days, produce antibody after result display immunity, immunizing dose is that 0.3mL, 0.4mL, 0.5mL immunity latter 14 days antibody positive rate reach 100%, and immunity is attacked malicious protection ratio in latter 28 days and reached 100%, and control group chicken is all dead after attacking poison.Therefore, the minimum immune dosage of this inactivated vaccine is 0.3mL/ (table 6).
The protest test of table 6 Different vaccine dosage
Similar immune effect of vaccine comparison test:
Get 21 age in days SPF chicken 70, be divided into 4 groups, A, B, C group is immune group, and A, B component Wei the strain of infectious bursa of Fabricius inactivated vaccine VNJ0Z, HQ immune group of Ministry of Agriculture's approved, and C group is IBDV-LY23 strain inactivated vaccine immune group, often organizes 20, D group 10.A, B group carries out immunity according to product description, C group every chicken nape portion subcutaneous inoculation 0.5ml, D group every aseptic PBS of chicken nape portion subcutaneous inoculation 0.5ml.Latter 7,14,21 days of immunity, blood sampling separation of serum, measures chicken infectivity bursa of Fabricius virus antibody, and immunity uses virulent IBD/SD/LY/2014 (containing 10 after latter 21 days
4eLD
50/ only) virus liquid per os attacks poison, Continuous Observation 72 hours, record morbidity and death condition, all slaughtered after 72 hours, checks fabricius bursa pathology.Result shows, and IBDV-LY23 strain inactivated vaccine group, antibody titers all higher than similar vaccine group, and well can resist the attack (table 7) of virulent epidemic strain.
The similar immune effect of vaccine comparison test of table 7
Immune duration measures:
Get 21 age in days SPF chicken 150,120 inoculation IBDV-LY23 strain inactivated vaccines, only, 30 is negative control to 0.5mL/, inoculation 0.5mL sterilizing PBS.The rear separation of serum of taking a blood sample respectively for 14,28,42,56,70,98 days of immunity, measures chicken infectivity bursa of Fabricius virus antibody, gets 10 with virulent IBD/SD/LY/2014 (containing 10 after blood sampling
4eLD
50/ only) virus liquid per os attacks poison, measures protection ratio.Result shows, immunity latter 14 days antibody positive rate reach 85.00%, and virulent is attacked malicious protection ratio and reached 10/10; after 28 days; antibody positive rate reaches 93.30%, attacks malicious protection ratio and reaches 10/10, and immunity latter 42 days antibody positive rate reach 96.67%; attack malicious protection ratio and reach 10/10; may persist to 70 days, latter 98 days of immunity, antibody titers, positive rate and protection ratio decline to some extent; control group chicken all falls ill, and all occurs typical fabricius bursa infection symptoms.Result shows, and the IBDV-LY23 strain inactivated vaccine antibody extended period is long, can provide effective protection, can resist the attack (table 8) of IBD/SD/LY/2014 virulent.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
。
Claims (7)
1. chicken infectivity bursa of Fabricius virus IBDV-LY23 strain, it is Very virulent infectious bursal disease virus DF-1 cell adapted strain, and deposit number is CGMCCNo.11594.
2. described in claim 1, the application in bursal disease vaccine is being prepared in chicken infectivity bursa of Fabricius virus IBDV-LY23 strain.
3. application according to claim 2, is characterized in that, is mixed by a certain percentage by the inactivation of viruses liquid of IBDV-LY23 strain and obtain bursal disease vaccine with adjuvant.
4. application according to claim 3, is characterized in that, the deactivation of described IBDV-LY23 strain beta-propiolactone.
5. the application according to claim 3 or 4, is characterized in that, described adjuvant is MONTANIDEISA71VG.
6. application according to claim 5, is characterized in that, by IBDV-LY23 strain beta-propiolactone deactivation, is mixed by inactivation of viruses liquid with MONTANIDEISA71VG according to weight ratio 3:7, obtains described vaccine through emulsification.
7. prevent a vaccine composition for infectious bursal disease, it is characterized in that, the effective constituent of described vaccine composition comprises the Very virulent infectious bursal disease virus DF-1 cell adapted strain according to claim 1 after deactivation.
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| CN109207436B (en) * | 2017-07-07 | 2022-02-18 | 乾元浩生物股份有限公司 | Group I type 4 avian adenovirus strain and application thereof |
| CN111235117A (en) * | 2020-02-13 | 2020-06-05 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Chicken infectious bursal disease virus classical strain naturally adapting to in vitro cell culture and application thereof |
| CN113061583A (en) * | 2021-03-30 | 2021-07-02 | 天津瑞普生物技术股份有限公司 | Method for culturing avian encephalomyelitis virus by using primary cells of chicken bursa of Fabricius |
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