CN113755454B - 8a type avian adenovirus strain, inactivated vaccine, preparation method and application thereof - Google Patents
8a type avian adenovirus strain, inactivated vaccine, preparation method and application thereof Download PDFInfo
- Publication number
- CN113755454B CN113755454B CN202111208145.7A CN202111208145A CN113755454B CN 113755454 B CN113755454 B CN 113755454B CN 202111208145 A CN202111208145 A CN 202111208145A CN 113755454 B CN113755454 B CN 113755454B
- Authority
- CN
- China
- Prior art keywords
- strain
- virus
- adenovirus
- avian
- lmh
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/20—Antivirals for DNA viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/525—Virus
- A61K2039/5252—Virus inactivated (killed)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/10011—Adenoviridae
- C12N2710/10211—Aviadenovirus, e.g. fowl adenovirus A
- C12N2710/10221—Viruses as such, e.g. new isolates, mutants or their genomic sequences
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/10011—Adenoviridae
- C12N2710/10211—Aviadenovirus, e.g. fowl adenovirus A
- C12N2710/10234—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/10011—Adenoviridae
- C12N2710/10211—Aviadenovirus, e.g. fowl adenovirus A
- C12N2710/10251—Methods of production or purification of viral material
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention relates to the technical field of biological products for livestock, and particularly discloses an 8 a-type avian adenovirus strain, an inactivated vaccine, a preparation method and application thereof. The strain belongs to group I E type 8a, is named as DK02 strain, and is preserved in China Center for Type Culture Collection (CCTCC) NO: V202170. Experiments show that the DK02 strain provided by the invention is suitable for suspending LMH cells (chicken liver cancer cells), and has the advantages of high toxicity, strong pathogenicity and good immunogenicity. The invention also provides an application of the DK02 strain in preparing vaccines for preventing inclusion body hepatitis. The invention provides an inactivated vaccine of avian I group adenovirus serum 8a type, which is used for preventing inclusion body hepatitis. After the chicken is immunized, the chicken 'inclusion body hepatitis' caused by the 8a adenovirus of the poultry I group in recent years can be well prevented, the toxicity attack protection rate is high, the safety is high, the antibody is produced quickly, and the efficacy is stable.
Description
Technical Field
The invention relates to the technical field of biological products for livestock, in particular to an I group E type 8a avian adenovirus strain, an inactivated vaccine, a preparation method and application thereof.
Background
Avian adenoviruses (fowladeoviruses) belong to the genus avirus of the family adenoviridae and are double-stranded DNA viruses without capsids. Avian adenoviruses can be divided into three groups: avian group I adenoviruses share a common group antigen and are classified into 5 types (FAV A-E) based on the determination of a cross neutralization assay, including 12 serotypes, representing strains with serotype 4 avian group I adenoviruses; group II FAV includes turkey hemorrhagic enteritis virus, pheasant marble spleen virus and avian splenomegaly virus; group III FAV contains only egg drop syndrome virus (EDSV-76). The avian group I adenovirus is specifically divided into 5 genotypes such as A, B, C, D, E and the like, and is divided into 1, 2, 3, 4, 5, 6, 7, 8a, 8b, 9, 10 and 11 types according to serotypes; the genotypes and serotypes correspond to A (1), B (5), C (4, 10), D (2, 3, 9, 11), E (6, 7, 8a, 8B). The avian adenovirus is recessive in early infection, has lower death rate, and shows different pathogenicity when reaching late infection, and clinically, diseases caused by the avian adenovirus mainly comprise myogastric erosion, inclusion body hepatitis, pericardial effusion-hepatitis syndrome and the like. Since 2012, epidemic situations in China are on an ascending trend.
The serotype 8 avian adenoviruses are divided into 8a and 8b serotypes, and are mainly infected with various chickens of 3-8 weeks of age, with overall mortality typically being below 10%, but sometimes up to 30%. The characteristic pathological changes of the disease are inclusion body hepatitis, which is characterized in that the liver is faded to light brown to yellow, is crisp and fragile, swells and is steatosis, the surface of the disease is provided with bleeding points or bleeding spots with different degrees, and the disease is accompanied by focal necrosis, and the inclusion body with alkalophilicity or acidophilicity can be seen in the nucleus of the liver cell, and the edge of the disease is larger and clear and is round or irregular in shape. In some cases, spleen is enlarged and bleeding occurs. The histopathological characteristic changes were mainly: liver cells undergo lipogenesis accompanied by vacuolation, rupture of some cells, atrophy of liver cells, etc. In most cases, the hepatic cable disintegrates and the hepatocytes exhibit pronounced vacuolation, and stained sections show that pronounced vacuolation of hepatocytes is caused by steatosis. The focal areas of degeneration and necrosis are randomly distributed throughout the liver tissue. Most hepatocytes can see varying degrees of atrophy, nuclear lysis, and nuclear lysis and leave suspended cells composed of one or more large vacuoles. Part of the hepatocytes were seen with nuclear swelling and chromatin marginalization. Cowdry type A nuclear inclusion bodies are found in many denatured hepatocytes. The 12 serotypes of the avian group I adenovirus can cause the occurrence of inclusion body hepatitis, but the main factors are FAdV-2, FAdV-8a, FAdV-8b and FAdV-11, and the pathogenicity of the avian group I adenovirus is different due to the different serotypes and strains. Inclusion body hepatitis starts to appear in the layer groups of 10-20 weeks old in 2012 in China, the incidence rate of the disease in China in recent years is in an ascending trend, and great economic loss is caused for the chicken industry in China.
The avian group I adenovirus can be transmitted vertically or horizontally, but mainly vertically. The virus can be transmitted vertically in the form of hatching eggs, and in addition, the virus also exists in chick embryo kidney cells and chick embryo liver cells. And a report on the detection of a virus in chickens with a chicken house. Although viruses can be separated from 1-day-old chicks, usually, the virus expelling of the sick chicks begins after 3 weeks of infection, the peak of the virus expelling of the broilers is 4-6 weeks old, and the egg-laying hens are 5-9 weeks old. The virus is mainly horizontally transmitted by directly contacting feces of the sick chickens, but in a small range, the virus can be slowly transmitted by air for a long time.
The chicken inclusion body hepatitis caused by the adenovirus of the poultry I group causes great economic loss to poultry farming industry of various countries, and the immune effect of most vaccine chick embryos or tissue inactivated vaccine used in the current market is not ideal. The serogroup of the avian I group adenovirus is more, the virus characteristics and pathogenicity of the avian I group adenovirus are greatly different, and the difficulty of developing and applying the vaccine is increased. E8a is a recently popular serotype, lacking a vaccine with pertinence.
Disclosure of Invention
The invention aims to provide a serogroup 8a adenovirus strain of an avian group I suitable for LMH suspension cell culture and application thereof. Separating, identifying and screening FAdV-8a virus strains which are infected and epidemic by Chinese chicken flocks, culturing the FAdV-8a virus strains by LMH suspension cells to obtain a vaccine strain with excellent antigenicity, and preparing corresponding vaccines has important significance for preventing the infection of 8a adenovirus of the poultry I flocks and the infection of other subtype avian adenoviruses.
The technical scheme for realizing the purpose of the invention is as follows:
firstly, providing an avian adenovirus serum 8a DK02 strain which is preserved in China center for type culture collection, wherein the address is: china, university of Wuhan, having a preservation date of 2021, 09, 17 days, and a preservation number of: CCTCC NO: V202170. The virus strain is identified as E in adenovirus I group adenovirus of avian adenovirus, the serotype is 8a, and the virus can be cultured by the allantoic cavity, chorioallantoic membrane and 6-day-old chick embryo yolk sac of 9-10-day-old chick embryos, and can also proliferate on the LMH cells of the chick liver cancer cells; the avian adenovirus DK02 strain is separated from liver tissues of dead chickens suffering from avian inclusion body hepatitis-pericardial hydrocephalus syndrome, is obtained by separating and proliferating chick embryos, can be adaptively proliferated on chicken liver cancer cells through cloning and passage adaptation, has strong strain proliferation capability and good immunogenicity, and is suitable for producing inactivated vaccines as vaccine strains.
The biological vaccine prepared by the strain contains inactivated antigen of avian I group adenovirus DK02 strain cultured by suspension chicken liver cancer cells, and the content of the inactivated antigen is more than or equal to 10 before inactivation 8.5 TCID 50 /0.1ml。
The strain is obtained by plaque cloning and purification, and the specific preparation method comprises the following steps:
(1) Selecting LMH adherent cells fully paved with a monolayer, discarding the original culture solution, adding a cell maintenance solution containing 1% of avian adenovirus DK02 strain virus solution, and placing at 37 ℃ and 5% of CO 2 Culturing, when cytopathy reaches more than 80%, freezing and thawing for 2 times, collecting virus liquid, namely F1 generation virus, and carrying out domestication culture on F1 generation virus on LMH adherent cells until F5 is passed;
(2) The F5 generation poultry glandVirus DK02 strain virus solution was 10-fold gradient diluted to 10 with DMEM -5 ～10 -9 Inoculating LMH cells growing into compact monolayer, adsorbing for 1 hr, removing liquid, adding a first layer of nutrient agar with thickness of about 3mm containing low melting point agarose with final concentration of 1% and DMEM containing 2% foetal calf serum, and placing CO 2 Incubator, 37 ℃,5% CO 2 After culturing for 48h under inverted condition, spreading a second layer of nutrient agar containing 1 XDMEM with final concentration of 1% low melting point agarose and 2% foetal calf serum at 37deg.C and 5% CO when cells are diseased 2 Culturing in the dark;
(3) Sucking out the formed plaque by using a dripper, putting the formed plaque and agarose into 1.0ml of serum-free DMEM, repeatedly freezing and thawing for 3 times to fully release the virus, inoculating new blank LMH cells, carrying out virus proliferation, repeating cloning for three times, randomly selecting five plaques of fourth-generation clone, namely F9 generation virus, culturing, measuring the virus content, respectively selecting one strain with the highest virus content, and continuously passaging through the LMH cells to obtain the purified virus liquid of the avian I group adenovirus DK02 strain.
The strain is suitable for culturing adherent and suspended LMH cells, and the method comprises the following steps:
(1) Preparation of adherent LMH cells and virus inoculation and harvesting: selecting LMH adherent cells with good growth vigor, discarding the original culture solution, adding cell maintenance solution containing 1% of avian adenovirus DK02 strain virus solution, standing at 37deg.C and 5% CO 2 Culturing, when cytopathy reaches more than 80%, freezing and thawing for 2 times, and harvesting virus liquid, namely DK02 strain virus liquid suitable for culturing the adherent LMH cells;
(2) Suspension LMH cell preparation and virus inoculation and harvesting: selecting suspended LMH cells with initial density of about 200 ten thousand, properly diluting basic strain DK02 of avian adenovirus obtained by propagating the adherent LMH cells by DMEM, inoculating the basic strain DK02 into the suspended LMH cells, shaking and culturing at 37 ℃ and 120rpm for 72-96 hours, freezing and thawing for 2 times, harvesting the virus liquid, sub-packaging and annotating the generation times of the virus seed and the harvesting time;
an inactivated vaccine for group I, E, type 8a avian adenovirus, the antigen of said vaccine being derived from the avian adenovirus DK02 strain. The preparation method comprises the following steps:
preparing an oil phase:
taking 95 parts of mineral oil and 1 part of aluminum stearate, uniformly mixing in an oil phase preparation tube, heating to 75-85 ℃, adding 5 parts of span 80 again, maintaining for 20-40 minutes when the temperature reaches 110-120 ℃, and cooling to finish oil phase preparation;
preparing an aqueous phase:
inoculating the DK02 strain with suspended LMH cells, culturing for 72-96 h, and obtaining a culture as virus liquid; taking 95 parts of inactivated avian adenovirus serum 8a type virus antigen liquid, and 5 parts of sterilized Tween 80, and fully and uniformly mixing;
emulsification:
and (3) taking 2 parts of oil phase and 1 part of water phase, and placing the oil phase and the water phase into an emulsifying tank for stirring to obtain the inactivated vaccine.
The beneficial effects of the invention are as follows: the group I8 a type avian adenovirus strain DK02 provided by the invention is a natural isolate, is obtained through plaque purification and screening, and is cultured into serum 8a type avian adenovirus with stable titer through optimization of culture conditions such as optimal inoculation dosage, optimal harvest time and the like, and has good immunogenicity and specificity and high yield. Meanwhile, the vaccine has a preventive effect on chicken inclusion body hepatitis caused by the adenovirus serum 8a of the poultry I group, and after the adenovirus is prepared into an inactivated vaccine, researches on the production process, the safety, the virus attack protection effect, the immunity dosage and the like of the vaccine show that the vaccine has good safety, high virus attack protection rate and stable immunity effect.
Drawings
FIG. 1 is a diagram of the present invention for PCR identification of avian group I adenoviruses from a clinically isolated sample of avian adenovirus DK02 strain, wherein M: dnastar DL2501,1: avian group I adenovirus negative control, 2: positive control of fowl I group adenovirus, 3-5: samples were isolated clinically.
FIG. 2 is a genetic phylogenetic tree of the avian adenovirus Hexon genes.
FIG. 3 shows the results of a plaque clone purification assay, wherein white spots are formed by the avian group I adenovirus DK02 strain used in the examples of the present invention.
Detailed Description
Preferred embodiments of the present invention will be described in detail below with reference to examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention may be made by those skilled in the art without departing from the spirit and scope of this invention. The experimental methods used in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
EXAMPLE 1 isolation, purification and identification of strains
Isolation of avian adenovirus DK02 strain
Collecting liver and spleen tissues of a sick chicken with typical symptoms and pathological changes, shearing and grinding, weighing, adding sterilized PBS (phosphate buffer solution) according to the weight ratio of 1:3, centrifuging at the temperature of 4 ℃ and 6000r/min for 10min, centrifuging at the temperature of 4 ℃ and 8000r/min for 10min, taking supernatant, transferring the supernatant into another sterile 1.5ml centrifuge tube, adding diabody (the final concentration is 2000IU/ml, 2mg/ml of streptomycin), acting at the temperature of 37 ℃ for 1h, and inoculating 5 SPF chick embryos of 5 days old into yolk sacs respectively, wherein the concentration is 0.2 ml/chicken embryo. Discarding the dead chick embryo within 24 hours, observing for 1 time every 6 hours, collecting allantoic fluid and embryo bodies of the dead chick embryo, taking out all chick embryo allantoic fluid and embryo bodies until 216 hours, mashing tissues, diluting with sterile PBS containing green streptomycin for 2 times, reserving samples for detection, and preserving virus isolates at-70 ℃.
Hexon gene sequence determination and genetic evolution analysis
Sample nucleic acid is extracted, a primer is designed according to the published FAV-I hexon gene sequence to carry out PCR identification on genes, the band size is about 2800bp (figure 1), and positive samples are subjected to clone sequencing. The analysis of the genetic evolution tree constructed on the sequencing result shows that the isolated strain has the closest relationship with the avian adenovirus I subgroup 8a and has far relationship with other serotypes of I subgroups such as serotype 4 and the like (figure 2). The separate strain is used for preparing the vaccine, so that the vaccine has a good epidemic prevention effect on the E8a type epidemic strain of the avian I group adenovirus.
Virus cell acclimation and plaque clone purification
Selecting LMH adherent cells with good growth vigor, discarding the original culture solution, adding cell maintenance solution containing 1% virus solution, standing at 37deg.C and 5% CO 2 Culturing, freezing and thawing for 2 times when cytopathy reaches more than 80%, and collecting virus liquid, namely F1 generation virus. And (3) passaging the F1 generation virus to F5 on LMH adherent cells, and performing domestication culture.
Diluting F5 virus solution with DMEM 10 times gradient to 10 -5 ～10 -9 Inoculating into LMH cells growing into compact monolayer, adsorbing for 1 hr, removing liquid, adding first layer of nutrient agar (DMEM with final concentration of 1% low melting point agarose and 2% fetal bovine serum) with thickness of about 3mm, adding CO 2 Incubator, 37 ℃,5% CO 2 Culturing under inversion for 48 hr (when the cells have lesions), spreading a second layer of nutrient agar (1 XDMEM with final concentration of 1% low melting point agarose and 2% foetal calf serum), 37deg.C, 5% CO 2 And culturing in the dark. After plaque formation (FIG. 3) was aspirated with a dropper, placed in 1.0ml serum-free DMEM along with agarose, repeatedly frozen and thawed 3 times to allow sufficient release of virus, inoculated with new blank LMH cells, and virus propagated, and cloned repeatedly three times. Randomly selecting five plaques of fourth-generation clone, namely F9-generation virus, culturing, measuring the virus content, respectively selecting one strain with the highest virus content, continuously passaging through LMH cells, taking F11-generation virus as a virus for a subsequent virus attack experiment, and taking F15-generation virus as a basic seed virus for upper suspension cell culture.
Identification of avian group I adenovirus DK02 strain virus species
(1) Sterility and mycoplasma assays
The test is carried out according to the annex of the current Chinese veterinary pharmacopoeia, and after the DK02 strain F15 generation basic seed virus sample is inoculated, the bacteria and mycoplasma grow.
(2) Virus content assay
10 times serial dilution of DK02 strain F15 virus liquid is carried out, and 10 times serial dilution is carried out -6 、10 -7 、10 -8 、10 -9 、10 -10 Five dilutions were inoculated with a monolayer of LMH cells on a 96-well cell plate, and incubated at 37 ℃ for 72 hours. After 72 hours, cytopathy was observed from well to wellThe virus content was calculated according to the Reed-Muench method, with the typical lesions of the inoculated cells being judged as infection. The results show that: DK02 strain F15 strain virus content of 10 7.85 TCID 50 /0.1ml。
(3) Exogenous virus assay
The method is carried out by adopting a chicken test method, virus liquid is emulsified after being inactivated by formaldehyde, 14-day-old SPF chicken is inoculated by an intramuscular injection way, and the inoculation is repeated for 1 time again by the same method after 21 days. Blood was collected 42 days after the first inoculation, and serum antibody monitoring of the relevant pathogen was performed. The result shows that the DK02 strain virus liquid has no foreign virus pollution.
TABLE 1 serological test results of DK02 exogenous Virus strain
| Inspection of pathogens | Inspection method | DK02 strain | Positive control | Negative control |
| Avian group III adenovirus (with hemagglutination) | HI | - | + | - |
| Avian influenza A virus | AGP/HI | - | + | - |
| Avian leukemia virus | ELISA | - | + | - |
| Avian encephalomyelitis virus | ELISA | - | + | - |
| Avian reticuloendotheliosis virus | IFA/ELISA | - | + | - |
| Fowl pox virus | AGP/clinical observations | - | + | - |
| Avian reovirus | AGP/ELISA | - | + | - |
| Marek's disease virus of chicken | AGP | - | + | - |
| Newcastle disease virus | HI | - | + | - |
| Infectious bronchitis virus | HI/ELISA | - | + | - |
| Infectious laryngotracheitis virus of chicken | Neutralizing antibodies/ELISA | - | + | - |
| Infectious bursal disease virus | AGP/ELISA | - | + | - |
Note that: "-" represents negative, "+" represents positive.
(4) Minimum pathogenic amount determination
Taking the F11 substitution virus of the avian adenovirus DK02 strain for a virus attack test. The 10-day-old SPF chickens were each intramuscular injected with different doses of DK02 strain virus and observed for l4 days. The results showed that both morbidity and mortality of the test chickens were concentrated within 7 days after challenge, after which no additional morbidity was observed for 14 days. 10 6.0 TCID 50 、10 5.0 TCID 50 The attack rate is 10/10, and the death rate is 8/10-10/10; 10 4.0 TCID 50 The attack rate is 8/10, and the death rate is 8/10;10 3.0 TCID 50 The attack rate is 7/10, and the death rate is 6/10; PBS control chicken 10/10 was normal. Experiments show that the minimum pathogenic amount is 10 4.0 TCID 50 . The chicken with the dead disease can be seen to have yellowish liver, swelling, crisp and fragile liver, swollen kidney and small amount of pericardial effusion after dissecting. The control group had no clinical symptoms. The results are detailed in Table 2.
TABLE 2 results of pathogenicity tests on SPF chickens with different amounts of challenge agent
Disease criteria: (1) clinical symptoms: mental depression, coarse and disordered feathers, eye closure, squat and the like, and serious death; (2) and (3) performing dissecting and pathological changes: the pathological changes of the dead or non-dead chicken within 2-3 days after clinical symptoms appear, such as yellowish liver, swelling, crisp and fragile texture, enlarged kidney, small amount of pericardial effusion and the like are found by the section inspection. (3) Nucleic acid detection: the fowl I group adenovirus 8a type nucleic acid in liver is positive. Meets the criterion of any item as pathogenesis.
(5) Immunogenicity determination
Inoculating DK02 strain F15 strain virus, inoculating LMH suspension cells with good growth, and harvesting virus liquid to prepare an inactivated vaccine, wherein the specific method is as follows: mixing white oil and span-80 at a volume ratio of 94:6 to obtain vaccine oil phase. And inactivating the virus liquid of the avian adenovirus DK02 strain by formaldehyde to obtain an inactivated virus liquid. Mixing the inactivated virus solution and tween-80 at a volume ratio of 96:4, and taking the mixture as vaccine water phase. Mixing the oil phase and the water phase at a volume ratio of 2:1 to obtain the inactivated vaccine.
The inactivated vaccine prepared by the F15 virus seed is respectively immunized with 10 SPF chickens of 7 days old under the neck skin according to 0.3 ml/feather, and 10 non-immunized chickens are taken as blank control. 21 days after immunization, all test chickens were sampled and serum was isolated and subjected to an agar diffusion test by reference to the method in the current "animal biological product test protocol". Meanwhile, the virus attack protection test of the avian adenovirus DK02 strain (F11 generation) is carried out on chickens in an immune group and a control group.
Toxicity attack protection test: the avian adenovirus DK02 strain (F11 generation) is used for counteracting the virus, and each chicken is injected with 0.2ml (more than or equal to 10) of muscle 5.0 TCID 50 0.1 ml), observing the state of the chicken flock every day within 10 days after the virus attack, and judging that the chicken flock is unprotected if the chicken flock is ill; if the tested chicken is only detoxified and does not attack the disease in the observation period, the tested chicken is judged to be protected. The result shows that the positive rate of the immune group serum agave-expanded antibody is up to 10/10 after the inactivated vaccine prepared by the F15 virus seed is immunized according to the dosage of 0.3 ml/feather dose for 21 days; the test result of the virus attack protection of the avian adenovirus DK02 strain (F11 generation) shows that all the virus attack chickens in the inactivated vaccine immune group prepared by the F15 generation virus seed are protected, and all the virus attack chickens in the control group are ill.
TABLE 3 determination of immunogenicity of DK02 strains
| Group of | Age of day | Immunization pathway | Immunity number | Immunization dose | Antibody Positive Rate | Antidote amount | Protection rate | Incidence of disease |
| DK02 strain | 7d | Subcutaneous tissue | 10 | 0.3ml | 10/10 | 0.2ml | 10/10 | 0/10 |
| Control | 7d | Subcutaneous tissue | 10 | / | 0/10 | 0.2ml | / | 10/10 |
Disease criteria: (1) clinical symptoms: mental depression, coarse and disordered feathers, eye closure, squat and the like, and serious death; (2) and (3) performing dissecting and pathological changes: the pathological changes of the dead or non-dead chicken within 2-3 days after clinical symptoms appear, such as yellowish liver, swelling, crisp and fragile texture, enlarged kidney, small amount of pericardial effusion and the like are found by the section inspection. (3) Nucleic acid detection: the fowl I group adenovirus 8a type nucleic acid in liver is positive. Meets the criterion of any item as pathogenesis.
The research result of biological characteristics of the avian adenovirus DK02 strain shows that the virus is an I group 8a epidemic strain, can proliferate on LMH adherent cells and LMH suspension cells at high titer, has good immunogenicity, and can effectively resist the attack of homologous viruses. Therefore, the strain can be used as a candidate strain of the avian adenovirus disease and used for developing an inactivated vaccine.
EXAMPLE 2 preparation of group I serogroup 8a avian adenovirus DK02 strain inactivated vaccine
DK02 Strain antigen preparation
Foundation virus of avian adenovirus DK02 strain F15The venom is properly diluted by DMEM and inoculated into suspended LMH cells with the initial density of about 200 ten thousand, and after shaking culture is carried out for 72-96 hours at 37 ℃ and 120rpm, the virus liquid is harvested, split charging is carried out to note the virus seed generation time, the harvesting time and the like, and the sample is reserved for detection. The sample to be detected is subjected to aseptic inspection according to the annex of the current Chinese animal pharmacopoeia, mycoplasma inspection and exogenous virus inspection, no bacteria, fungi, mycoplasma and exogenous virus pollution is needed, and the virus content before inactivation is more than or equal to 10 per 0.1ml virus liquid content 8.5 TCID 50 . The harvested virus liquid is preserved at 2-8 ℃ before inactivation for no more than 3 days.
DK02 strain virus culture was inactivated with 0.2% formaldehyde at 37℃for 24 hours. After the inactivation is finished, sampling is carried out for sterile inspection and inactivation inspection, and the sample is preserved at the temperature of 2-8 ℃ for no more than 7 days.
2. Inspection of semifinished products
(1) Sterile test, according to the appendix of the current Chinese animal pharmacopoeia, the inactivated DK02 strain is subjected to sterile test and should grow aseptically.
(2) Inactivation test
Diluting the inactivated virus solution by 10 times, inoculating LMH cells (24-well cell plate) with good growth vigor into 4 wells, adding maintenance solution to 1ml per well, setting chicken liver cancer cells as blank control, placing at 37deg.C and CO 2 Culturing in an incubator for 120 hours. No cytopathy appears in the cell control wells and the sample wells. And (3) carrying out blind transfer for 1 generation after repeated freeze thawing on the harvested culture, and continuing to culture for 120 hours according to the method, wherein the inactivation is judged to be complete when cytopathy does not appear in the sample hole.
3. Vaccine preparation
3 batches of inactivated vaccine were prepared according to the following steps:
(1) Preparing an oil phase: mixing 95 parts of mineral oil and 1 part of aluminum stearate uniformly in an oil phase preparation tube, heating to 80 ℃, adding 5 parts of span 80 again, maintaining for 30 minutes when the temperature reaches 115 ℃, and cooling to finish oil phase preparation;
(2) Preparing an aqueous phase: inoculating LMH cells to DK02 strain respectively, and harvesting the culture to obtain virus liquid after 80% of cells are diseased; inactivating the inactivated avian adenovirus DK02 strain virus liquid to obtain antigen liquid; taking 95 parts of antigen liquid, and sterilizing 5 parts of tween 80, and fully and uniformly mixing;
(3) Emulsification: 2 parts of oil phase and 1 part of water phase are taken, put into an emulsifying tank, stirred for 5 minutes at 3500r/min and 15 minutes at 8000r/min, and the emulsifying preparation is completed.
(4) Quantitatively split charging, capping and sealing, labeling, and storing at 2-8 ℃.
4. Vaccine product inspection
(1) Traits (3)
The appearance, formulation, viscosity and stability of 3 batches of vaccine were measured, respectively, and the results are shown in Table 4.
Table 4 3 test results of the characteristics of the inactivated seedlings
| Vaccine lot number | Appearance of | Dosage form | Viscosity (cP) | Stability of | Result determination |
| 20210401 | Milky homogeneous emulsion | Water-in-oil type | 25.47 | No water phase is separated out | Qualified product |
| 20210402 | Milky whiteHomogeneous emulsion | Water-in-oil type | 22.95 | No water phase is separated out | Qualified product |
| 20210403 | Milky homogeneous emulsion | Water-in-oil type | 23.10 | No water phase is separated out | Qualified product |
(2) Sterility testing
The filling amount of 3 batches of bigeminal seedlings is checked according to the method of the annex of the third part of the Chinese animal pharmacopoeia, and the results are not lower than the label marking amount of the bottle label, and are shown in Table 5 in detail.
Table 5 3 results of the test for the loading amount of the inactivated seedlings
| Vaccine lot number | Indicating capacity (ml/bottle) | Inspection results (ml) | Result determination |
| 20210401 | 250 | 251.2,251.7,251.4 | Qualified product |
| 20210402 | 250 | 252.0,251.3,252.1 | Qualified product |
| 20210403 | 250 | 252.0,251.1,251.5 | Qualified product |
(3) Sterility testing
The 3 batches of vaccines were subjected to sterility testing according to the method of the appendix of the current "third part of the Chinese veterinary pharmacopoeia", the results of which are all sterile grown, and the results of which are shown in Table 6 in detail.
Table 6 3 sterility test results for inactivated seedlings
(4) Safety test
1) Single dose vaccination safety test: 3 batches of DK02 inactivated vaccine were taken and injected into 7-day-old SPF chickens 10 by subcutaneous injection via the nape and leg muscles, respectively, each of which was 0.3ml. The spirit and diet of the test chickens were recorded in detail 28 days after injection, and 3, 4 and 3 were taken from each group for a split examination at 14, 21 and 28 days after injection, respectively, and the injection sites were observed. The test results are shown in Table 7.
TABLE 7 Single dose vaccination safety test results
2) Safety test for single dose repeat vaccination: taking 3 batches of group I serum 8a type avian adenovirus DK02 strain inactivated vaccine, injecting 10 SPF chickens of 7 days old into each batch of vaccine through subcutaneous neck and back and leg muscle respectively, injecting 0.3ml each, injecting 0.3ml again in the same way after 14 days, observing for 28 days, and recording the spirit and diet condition of the tested chickens in detail. And at 14, 21 and 28 days after the secondary injection, 3, 4 and 3 sections were taken from each group, respectively, and the injection sites were observed. The test results are shown in Table 8.
Table 8 safety test results for single dose repeat vaccination
3) Safety test for overdose inoculation: taking 3 batches of I-group serum 8 a-type avian adenovirus DK02 strain inactivated vaccine, and injecting 10 SPF chickens of 7 days old into each batch of vaccine through subcutaneous neck and back and leg muscles respectively, wherein each injection is 1.0ml; meanwhile, 20 chickens with the same conditions are arranged, and 1.0ml of sterile physiological saline is respectively injected into the subcutaneous neck and back and the leg muscles, and 10 chickens are injected in each way. 10 chickens were randomly drawn prior to injection and weighed. On day 14 post injection, 10 chickens from each vaccinated group were weighed along with 10 control chickens and the vaccinated group was compared for weight gain below the control group. At the same time, the spirit and diet of the test chickens were recorded in detail 28 days after injection, and 3, 4 and 3 sections were taken from each vaccinated group at 14, 21 and 28 days after injection, respectively, and the injection sites were observed. The test results are shown in Table 9.
TABLE 9 safety test results for overdose vaccination
(5) Efficacy test
Serological methods: 20 SPF chickens of 7 days old were taken, 10 of which were vaccinated subcutaneously in the neck or intramuscularly in the leg by 0.3m1, and 10 of which were used as controls. Each chicken was sampled 21 days after inoculation, serum was isolated, and the avian group I8 a adenovirus antibody was detected by a agar assay, at least 8 positive in the immunized group, and all negative in the control group. The test results are shown in Table 9.
Immune toxicity counteracting method:20 SPF chickens of 7 days old were taken, 10 of which were vaccinated subcutaneously in the neck or intramuscularly in the leg by 0.3m1, and 10 of which were used as controls. 21 days after inoculation, all immunized chickens and control chickens are intramuscular injected with the avian I group 8a adenovirus DK02 strain virus liquid (the virus content is more than or equal to 10) 5.0 TCID 50 0.1 ml), each 0.2ml, and the control chickens should be at least 8 at the onset and the immunized chickens should be at least 8 at the protection after 14 days of observation. The test results are shown in Table 10.
TABLE 10 efficacy test results
Disease criteria: (1) clinical symptoms: mental depression, coarse and disordered feathers, eye closure, squat and the like, and serious death; (2) and (3) performing dissecting and pathological changes: the pathological changes of the dead or non-dead chicken within 2-3 days after clinical symptoms appear, such as yellowish liver, swelling, crisp and fragile texture, enlarged kidney, small amount of pericardial effusion and the like are found by the section inspection. (3) Nucleic acid detection: the fowl I group adenovirus 8a type nucleic acid in liver is positive. Meets the criterion of any item as pathogenesis.
The test results show that the prepared inactivated vaccine of the avian I group 8a adenovirus DK02 strain is safe and effective. While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (7)
1. The 8a type avian adenovirus strain is characterized in that the strain is named as DK02 strain, the preservation number is CCTCC NO: V202170, wherein the virus strain is E types in avian adenovirus I group, and the serotype is 8a type.
2. The biologic vaccine prepared from the strain according to claim 1, wherein the vaccine comprises fowl cultured with suspension chicken liver cancer cellsGroup I adenovirus DK02 strain inactivated antigen with content of not less than 10 before inactivation 8.5 TCID 50 /0.1ml。
3. The method for preparing the strain according to claim 1, wherein the strain is obtained by plaque clone purification, and the specific preparation method comprises the following steps:
(1) Selecting LMH adherent cells fully paved with a monolayer, discarding the original culture solution, adding a cell maintenance solution containing 1% of avian adenovirus DK02 strain virus solution, and placing at 37 ℃ and 5% of CO 2 Culturing, when cytopathy reaches more than 80%, freezing and thawing for 2 times, collecting virus liquid, namely F1 generation virus, and carrying out domestication culture on F1 generation virus on LMH adherent cells until F5 is passed;
(2) Diluting F5-generation fowl adenovirus DK02 strain virus liquid with DMEM, inoculating to LMH cells grown into compact monolayer, adsorbing for 1 hr, removing liquid, spreading first layer of nutrient agar containing low melting point agarose with final concentration of 1% and DMEM containing 2% foetal calf serum, and placing CO 2 Incubator, 37 ℃,5% CO 2 After culturing for 48h under inverted condition, spreading a second layer of nutrient agar containing 1 XDMEM with final concentration of 1% low melting point agarose and 2% foetal calf serum at 37deg.C and 5% CO when cells are diseased 2 Culturing in the dark;
(3) Sucking out the formed plaque by using a dripper, putting the formed plaque and agarose into 1.0ml of serum-free DMEM, repeatedly freezing and thawing for 3 times to fully release the virus, inoculating new blank LMH cells, carrying out virus proliferation, repeating cloning for three times, randomly selecting five plaques of fourth-generation clone, namely F9 generation virus, culturing, measuring the virus content, respectively selecting one strain with the highest virus content, and continuously passaging through the LMH cells to obtain the purified virus liquid of the avian I group adenovirus DK02 strain.
4. The method of preparing a strain according to claim 1, characterized in that the method comprises the steps of:
(1) Preparation of adherent LMH cells and virus graftingSeed and harvest: selecting LMH adherent cells with good growth vigor, discarding the original culture solution, adding cell maintenance solution containing 1% of avian adenovirus DK02 strain virus solution, standing at 37deg.C and 5% CO 2 Culturing, when cytopathy reaches more than 80%, freezing and thawing for 2 times, and harvesting virus liquid, namely DK02 strain virus liquid suitable for culturing the adherent LMH cells;
(2) Suspension LMH cell preparation and virus inoculation and harvesting: and (3) properly diluting the basic strain of the avian adenovirus DK02 obtained by the proliferation of the adherent LMH cells by using DMEM, inoculating the basic strain of the avian adenovirus DK02 into the suspended LMH cells, shaking and culturing at 37 ℃ and 120rpm for 72-96 hours, freezing and thawing for 2 times, harvesting the virus liquid, and subpackaging and annotating the number of virus generations and the harvesting time.
5. Use of a strain according to claim 1 for the preparation of a veterinary vaccine for the prevention of inclusion body hepatitis caused by avian group 8a adenoviruses.
6. An inactivated vaccine for group I, E, type 8a avian adenovirus, wherein the antigen of said vaccine is from the avian adenovirus DK02 strain of claim 1.
7. The inactivated vaccine according to claim 6, wherein the inactivated vaccine preparation method comprises the steps of:
preparing an oil phase:
taking 95 parts of mineral oil and 1 part of aluminum stearate, uniformly mixing in an oil phase preparation tube, heating to 75-85 ℃, adding 5 parts of span 80 again, maintaining for 20-40 minutes when the temperature reaches 110-120 ℃, and cooling to finish oil phase preparation;
preparing an aqueous phase:
inoculating the DK02 strain with suspended LMH cells, culturing 72-96 h, and obtaining a culture as virus liquid; taking 95 parts of inactivated avian adenovirus serum 8a type virus antigen liquid, and 5 parts of sterilized Tween 80, and fully and uniformly mixing;
emulsification:
and (3) taking 2 parts of oil phase and 1 part of water phase, and placing the oil phase and the water phase into an emulsifying tank for stirring to obtain the inactivated vaccine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111208145.7A CN113755454B (en) | 2021-10-18 | 2021-10-18 | 8a type avian adenovirus strain, inactivated vaccine, preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111208145.7A CN113755454B (en) | 2021-10-18 | 2021-10-18 | 8a type avian adenovirus strain, inactivated vaccine, preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113755454A CN113755454A (en) | 2021-12-07 |
| CN113755454B true CN113755454B (en) | 2023-07-07 |
Family
ID=78799603
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111208145.7A Active CN113755454B (en) | 2021-10-18 | 2021-10-18 | 8a type avian adenovirus strain, inactivated vaccine, preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113755454B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109207436A (en) * | 2017-07-07 | 2019-01-15 | 乾元浩生物股份有限公司 | One plant of 4 type aviadenovirus strain of I group and its application |
| CN111548996A (en) * | 2020-05-28 | 2020-08-18 | 山东诸子生物科技有限公司 | Poultry cancer cell serum-free suspension cell line and establishment method and application thereof |
| CN113185607A (en) * | 2021-03-22 | 2021-07-30 | 华南农业大学 | Preparation method of multivalent avian adenovirus egg yolk antibody |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA3177833A1 (en) * | 2009-12-15 | 2011-06-15 | University Of Saskatchewan | Vaccines for inclusion body hepatitis |
-
2021
- 2021-10-18 CN CN202111208145.7A patent/CN113755454B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109207436A (en) * | 2017-07-07 | 2019-01-15 | 乾元浩生物股份有限公司 | One plant of 4 type aviadenovirus strain of I group and its application |
| CN111548996A (en) * | 2020-05-28 | 2020-08-18 | 山东诸子生物科技有限公司 | Poultry cancer cell serum-free suspension cell line and establishment method and application thereof |
| CN113185607A (en) * | 2021-03-22 | 2021-07-30 | 华南农业大学 | Preparation method of multivalent avian adenovirus egg yolk antibody |
Non-Patent Citations (2)
| Title |
|---|
| Ⅰ群禽腺病毒及其疫苗研究进展;刘吉山;孙培姣;于雪;程志伟;沈志强;柳增善;肖跃强;;中国动物传染病学报(第01期);摘要 * |
| 我国Ⅰ群禽腺病毒主要流行血清型及其防控;尹燕博;;中国家禽(第03期);摘要 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113755454A (en) | 2021-12-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102220287B (en) | Avian infectious bronchitis cold adaptation attenuated vaccine strain and application thereof | |
| CN111000993B (en) | Bivalent inactivated vaccine for duck viral hepatitis and duck reovirus disease and preparation method thereof | |
| CN105582533B (en) | Avian influenza virus and avian adenovirus bivalent inactivated vaccine | |
| CN106282130A (en) | A kind of I group 4 type aviadenovirus, inactivated vaccine and preparation method thereof | |
| CN113491767A (en) | Triple inactivated vaccine for duck circovirus disease, novel duck reovirus disease and duck viral hepatitis and preparation method thereof | |
| CN109097340B (en) | Avian adenovirus, quadruple vaccine and preparation method thereof | |
| CN108465107B (en) | Duck type 2 adenovirus and Muscovy duck parvovirus disease combined inactivated vaccine | |
| CN111154730A (en) | Goose astrovirus variant strain | |
| CN109207436B (en) | Group I type 4 avian adenovirus strain and application thereof | |
| CN114395536B (en) | Avian adenovirus type 4, 8 and 11 trivalent vaccine and preparation method and application thereof | |
| CN109207437B (en) | Group I8 avian adenovirus strain and application thereof | |
| CN105802918B (en) | Chicken's infectious bronchitis nephritis strain and its vaccine composition, preparation method and application | |
| CN108939063B (en) | Muscovy duck triple inactivated vaccine | |
| CN109055320B (en) | Infectious bronchitis virus isolate and application thereof in vaccine preparation | |
| CN107523556A (en) | A kind of aviadenovirus strain, vaccine combination and its application | |
| CN116042537A (en) | Avian reovirus strain QYH2020-QD, virus liquid, preparation method and application | |
| CN113755454B (en) | 8a type avian adenovirus strain, inactivated vaccine, preparation method and application thereof | |
| CN114891753B (en) | Novel duck reovirus passaging attenuated strain and application thereof | |
| CN116286670A (en) | Novel duck reovirus and application thereof in preparation of inactivated vaccine and egg yolk antibody | |
| CN101716342B (en) | New castle disease and infectious bronchitis integrated inactivated vaccine and manufacture method thereof | |
| CN104195114A (en) | Avian pneumovirus and application thereof | |
| CN112063596A (en) | Pigeon paramyxovirus type 1 PPMV-1/BJ-C strain and application thereof | |
| CN102688487A (en) | Production method of newcastle disease (ND), infectious bronchitis (IB), infectious bursal disease virus (IBD) and viral arthritis (VA) four-joint inactivated vaccine | |
| CN111110839A (en) | Goose astrovirus bivalent inactivated vaccine for preventing gosling gout | |
| CN110079509A (en) | One kind I group of strain of 11d type aviadenovirus, inactivated vaccine and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |