CN115322972B - H9 subtype avian influenza virus isolate and application thereof - Google Patents
H9 subtype avian influenza virus isolate and application thereof Download PDFInfo
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- CN115322972B CN115322972B CN202210861893.3A CN202210861893A CN115322972B CN 115322972 B CN115322972 B CN 115322972B CN 202210861893 A CN202210861893 A CN 202210861893A CN 115322972 B CN115322972 B CN 115322972B
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Abstract
The application provides an H9 subtype avian influenza HF strain and a vaccine composition prepared from an inactivated antigen thereof, wherein the strain has good immunogenicity, can completely protect the existing H9 subtype avian influenza under the condition of low content, and can be used for preparing combined vaccine and composite vaccine with various other antigens.
Description
The application relates to a Chinese patent application with the name of an isolated strain of H9 subtype avian influenza virus and application thereof, which is a divisional application of Chinese patent application with the application number of 201910928612.X, the application date of 2019, 09 and 28.
Technical Field
The application relates to the field of animal virology, in particular to a method for separating, identifying and purifying an H9N2 avian influenza virus strain and an inactivated vaccine prepared by using the strain for preventing avian influenza.
Background
Avian Influenza (AI) is an acute, febrile, highly contagious Avian infectious disease caused by Avian influenza virus (Avian influenza virus, AIV), which is a single-stranded negative strand segmented RNA virus belonging to the genus influenza a, family orthomyxoviridae. The influenza a viruses can be clinically classified into highly pathogenic avian influenza viruses and low pathogenic avian influenza viruses according to the pathogenicity and virulence of the viruses, while the influenza a viruses can be classified into different subtypes according to the difference between the surface glycoproteins Hemagglutinin (HA) and Neuraminidase (NA), and currently there are 16 influenza virus HA subtypes and 10 influenza virus NA subtypes isolated in birds.
The low pathogenicity avian influenza viruses are various, but mainly take the subtype H9 as the main, wherein the subtype H9N2 avian influenza viruses are widely popular in China. The subtype virus has high morbidity and low mortality rate when the subtype virus simply infects chickens, only slight respiratory symptoms appear, and the laying rate and the hatching rate are reduced, but the subtype virus is easy to generate synergistic effect with other pathogenic microorganisms, and causes secondary infection, thereby causing high mortality rate of poultry groups, and having great harm to the poultry industry.
At present, vaccination is one of the main means for preventing and controlling H9N2 subtype avian influenza, but because viruses can generate variant strains through two ways of antigen drift and antigen transformation, the antigenicity of HA and NA is changed, and the immunization is failed. Especially in recent years, the condition that the poultry flock is infected with the H9N2 subtype avian influenza continuously occurs, even in immune flocks, and the phenomenon occurs because of the large genetic distance and antigenicity difference between the existing vaccine strain and the existing epidemic wild strain, so that the immunoprotection efficacy of the vaccine strain is reduced. Thus, there is a problem in that research and development of a vaccine for preventing H9 subtype avian influenza, which can provide effective cross protection against epidemic wild strains, is required.
Disclosure of Invention
In order to solve the existing technical problems, the invention provides an H9 subtype avian influenza virus isolate and an inactivated vaccine prepared by using the strain. The avian influenza virus isolate belongs to an H9 subtype, has good cross immunogenicity, and the inactivated vaccine prepared by the isolate can provide effective cross protection for novel H9 subtype avian influenza epidemic wild strains.
In order to achieve the purpose, the invention separates a strain of H9 subtype avian influenza HF strain from a certain pathogenic chicken farm in China, and the preservation number is CCTCC NO: v201941.
The HF strain has the following characteristics:
(1) The strain is an avian influenza virus H9N2 subtype strain.
(2) The strain can grow in chicken embryo with high titer, and the hemagglutinin titer is stabilized at 1:512.
(3) Half the infection amount (EID) of the strain on chick embryo 50 ) Up to 10 9.17 0.1ml, and the intravenous inoculation pathogenicity index (IVPI) is 0, which proves that the strain is a low pathogenicity strain.
(4) Cross hemagglutination inhibition experiments prove that the strain has good antigen correlation with the novel H9 subtype avian influenza epidemic wild strain.
Avian influenza virus (Subtype H9) HF Strain (Avian Influenza Virus (Subtype H9), strain HF) with a accession number cctccc NO: v201941, the preservation date is 2019, 06 and 19, and the preservation address is university of Wuhan and Wuhan in China.
The term subtype H9 avian influenza virus as used in the present invention is also referred to as subtype H9 avian influenza virus.
Similarly, the term avian influenza virus H9 subtype HF strain used in the present invention is also referred to as H9 subtype avian influenza virus HF strain.
The invention also relates to a vaccine composition, wherein the vaccine composition comprises an immunity amount of the H9 subtype avian influenza HF strain or a culture inactivated antigen thereof and a pharmaceutically acceptable carrier.
As one embodiment of the invention, the content of the inactivated antigen of the H9 subtype avian influenza HF strain or the culture thereof in the vaccine composition is more than or equal to 10 before inactivation 6.5 EID 50 /0.1ml。
The H9 subtype avian influenza HF strain or the culture inactivated antigen of the invention has good immunogenicity, and the content is only 10 6.5 EID 50 0.1ml also gives complete immunoprotection.
As a preferred embodiment of the present invention, in the vaccine composition of the present invention, the content of the inactivated antigen of the H9 subtype avian influenza HF strain or the culture thereof is 10 before inactivation 6.5 ～10 8.5 EID 50 /0.1ml。
As a more preferable embodiment of the invention, in the vaccine composition of the invention, the content of the inactivated antigen of the H9 subtype avian influenza HF strain or the culture thereof is 10 before inactivation 8.0 EID 50 /0.1ml。
As an embodiment of the present invention, in the vaccine composition of the present invention, the pharmaceutically acceptable carrier is an adjuvant, and the adjuvant includes: (1) White oil, alumina gel adjuvant, saponin, alfulidine, and DDA; (2) A water-in-oil emulsion, an oil-in-water emulsion, and a water-in-oil-in-water emulsion; or (3) a polymer of acrylic acid or methacrylic acid, a copolymer of maleic anhydride and an alkenyl derivative; and one or more of RIBI adjuvant system, block co-polymer, SAF-M, monophosphoryl lipid A, avridine lipid-amine adjuvant, E.coli heat labile enterotoxin, cholera toxin, IMS 1314, muramyl dipeptide, montanide ISA 206, gel adjuvant; preferably, the saponin is Quil A, QS-21, GPI-0100;
The adjuvant content is 5% -70% V/V.
Preferably, the adjuvant is a white oil adjuvant for preparing a water-in-oil emulsion;
the concentration of the adjuvant ranges from 5% to 70% V/V, preferably from 30% to 70%, more preferably 66% V/V.
The vaccine compositions of the present invention further comprise other pathogens or antigen combinations for use in preparing combination or combination vaccines against various diseases including H9 subtype avian influenza virus infection.
As an embodiment of the present invention, in the vaccine composition according to the present invention, the vaccine composition further comprises one or more of the following antigens: newcastle disease virus antigen, avian egg drop syndrome virus antigen, avian infectious bronchitis virus antigen, avian infectious bursal disease virus antigen, avian adenovirus antigen, avian reovirus antigen, escherichia coli antigen, parachicken bacillus antigen, synovial fluid cyst mycoplasma antigen, chicken mycoplasma antigen, pasteurella multocida antigen, marek's disease virus antigen, avian encephalomyelitis virus antigen, and avian infectious laryngotracheitis virus antigen.
As a preferred embodiment of the present invention, the vaccine composition according to the present invention further comprises one or more of the group consisting of: newcastle disease virus inactivated antigen, avian egg drop syndrome virus inactivated antigen, avian infectious bronchitis virus inactivated antigen, avian infectious bursal disease virus subunit antigen and avian adenovirus inactivated antigen.
As a more preferred embodiment of the present invention, the vaccine composition according to the present invention further comprises one or more of the group consisting of: the chicken newcastle disease virus N7a strain inactivated antigen, the avian egg drop syndrome virus HX strain inactivated antigen, the chicken infectious bronchitis virus M41 strain inactivated antigen, the chicken infectious bursal disease virus VP2 subunit antigen and the avian adenovirus FAV-HN strain inactivated antigen.
As a preferred embodiment of the present invention, in the vaccine composition of the present invention, the content of the inactivated antigen of the H9 subtype avian influenza virus HF strain or the culture thereof is 10 before inactivation 6.5 ～10 8.5 EID 50 0.1ml, wherein the content of the inactivated antigen of the newcastle disease virus is 10 before inactivation 8.0 ～10 9.0 EID 50 0.1ml, the content of the infectious bronchitis virus inactivated antigen is 10 before inactivation 6.0 ～10 7.0 EID 50 0.1ml, wherein the content of the inactivated antigen of the avian egg drop syndrome virus is 10 before inactivation 7.0 ～10 8.0 EID 50 0.1ml, wherein the content of VP2 protein of the infectious bursal disease virus is AGP titer 1:16-1:128, and the content of the inactivated antigen of the avian adenovirus is 10 before inactivation 5.0 ～10 8.0 EID 50 /0.1ml。
As a more preferable embodiment of the invention, in the vaccine composition of the invention, the content of the inactivation antigen of the H9 subtype avian influenza virus HF strain or the culture thereof is 10 before inactivation 8.0 EID 50 0.1ml, wherein the content of the inactivated antigen of the newcastle disease virus is 10 before inactivation 8.0 EID 50 0.1ml, the content of the infectious bronchitis virus inactivated antigen is 10 before inactivation 6.0 EID 50 0.1ml, wherein the content of the inactivated antigen of the avian egg drop syndrome virus is 10 before inactivation 7.0 EID 50 0.1ml, wherein the content of VP2 protein of infectious bursal disease virus is AGP titer 1:16, and the content of inactivated antigen of avian adenovirus is 10 before inactivation 6.5 EID 50 /0.1ml。
The vaccine compositions of the present invention may further incorporate additional agents into the compositions of the present invention.
As an embodiment of the invention, the veterinarily acceptable carrier includes a drug, an immunostimulant, an antioxidant, a surfactant, a colorant, a volatile oil, a buffer, a dispersant, a propellant, and a preservative; the immunostimulants include interferon-alpha, interferon-beta, interferon-gamma, granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), and interleukin 2 (IL 2).
Preferably, the immunostimulants include interferon-alpha, interferon-beta, interferon-gamma, granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), and interleukin 2 (IL 2).
To prepare such compositions, methods well known in the art may be used.
The invention also relates to application of the vaccine composition in preparing a medicament for preventing and/or treating H9 subtype avian influenza. The administration subjects for preparing the medicine for preventing and/or treating the H9 subtype avian influenza virus infection comprise chickens.
The invention also provides a preparation method of the H9 subtype avian influenza inactivated vaccine, which comprises the following steps:
the preparation of H9 subtype avian influenza virus HF strain virus liquid: inoculating AIV HF strain virus liquid to 10-day-old healthy SPF chick embryo through allantoic cavity, and harvesting 24-96 hours infected chick embryo liquid as H9 subtype avian influenza virus HF strain virus liquid;
inactivating the virus liquid of the H9 subtype avian influenza virus HF strain: taking the H9 subtype avian influenza virus HF strain virus liquid prepared in the step (1), adding 10 times diluted analytically pure formaldehyde solution to ensure that the final concentration is 0.1 percent (V/V), inactivating for 24 hours at 37 ℃, and shaking for 1 time at intervals of 4-6 hours;
preparing an oil phase in the step (3): mixing 94 parts by volume of white oil for injection and 6 parts by volume of span-80, adding 2 parts of aluminum stearate, heating while stirring until the mixture is transparent, and sterilizing under high pressure and cooling to obtain an oil phase solution; and
And (4) preparing an aqueous phase: taking the H9 subtype avian influenza virus HF strain virus liquid inactivated in the step (2), and adding sterile physiological saline to dilute the virus liquid into 10 8.5 EID 50 Uniformly mixing the diluted antigen and sterilized Tween-80 according to the volume ratio of 96:4 to obtain the water phase, wherein the antigen concentration is 0.1 ml;
step (5) vaccine emulsification: mixing 2 parts by volume of the oil phase in the step (3) and 1 part by volume of the water phase in the step (4), emulsifying uniformly, and adding 1% (W/V) of sulfur Liu Gongna solution before the emulsification is terminated to make the final concentration of the thimerosal sodium be 0.01% (W/V) so as to prepare the avian influenza inactivated vaccine.
The H9 subtype avian influenza virus strain and the inactivated vaccine prepared by the strain have the following advantages:
1. the H9 subtype avian influenza virus HF strain has good immunogenicity and can be used for preparing H9 subtype avian influenza vaccine.
2. After the prepared H9 subtype avian influenza inactivated vaccine is inoculated, high-titer antibodies can be generated, and the vaccine has good immunoprophylaxis effect on homologous and non-homologous H9 subtype avian influenza virus epidemic wild strains.
3. The adopted method for preparing the H9 subtype avian influenza inactivated vaccine is simple, safe and low in cost.
The avian influenza virus isolate belongs to the H9 subtype, is separated from an Anhui disease chicken farm, can be stably proliferated on chicken embryos, and can obtain virus liquid with high hemagglutination titer. The inactivated vaccine prepared from the H9 subtype avian influenza virus isolate with the characteristics can defend the attack of the novel H9 subtype avian influenza epidemic wild strain, has good cross immunogenicity, has better effect than the vaccine prepared from the strain in the prior art, and has good application prospect in the aspects of preventing and treating avian immune protection.
The invention also relates to an avian egg drop syndrome virus HX strain, the preservation number is CCTCC NO: v201942.
The avian egg drop syndrome virus HX strain has good immunogenicity, and the prepared inactivated vaccine can completely protect the current epidemic strain.
The invention also relates to a vaccine composition, wherein the vaccine composition comprises an immunity amount of inactivated antigen of the HX strain of the avian egg drop syndrome virus or a culture thereof and a pharmaceutically acceptable carrier; the inactivated antigen content of the HX strain or the culture of the avian egg drop syndrome virus is 10 7.0 ～10 8.0 EID 50 0.1ml, and the pharmaceutically acceptable carrier is an adjuvant.
As an embodiment of the present invention, in the vaccine composition of the present invention, the adjuvant comprises: the adjuvant comprises: (1) White oil, alumina gel adjuvant, saponin, alfulidine, and DDA; (2) A water-in-oil emulsion, an oil-in-water emulsion, and a water-in-oil-in-water emulsion; or (3) a polymer of acrylic acid or methacrylic acid, a copolymer of maleic anhydride and an alkenyl derivative; and one or more of RIBI adjuvant system, block co-polymer, SAF-M, monophosphoryl lipid A, avridine lipid-amine adjuvant, E.coli heat labile enterotoxin, cholera toxin, IMS 1314, muramyl dipeptide, montanide ISA 206, gel adjuvant; preferably, the saponin is Quil A, QS-21, GPI-0100;
The content of the adjuvant is 5-70% V/V;
preferably, the adjuvant is a white oil adjuvant for preparing a water-in-oil emulsion;
the concentration of the adjuvant ranges from 5% to 70% V/V, preferably from 30% to 70%, more preferably 66% V/V.
The avian egg drop syndrome virus HX strain inactivated antigen can be used as an oil emulsion vaccine prepared by the antigen to provide complete protection for chicken flocks.
The invention also relates to application of the vaccine composition in preparing medicines for preventing and/or treating the egg drop syndrome of poultry.
Drawings
FIG. 1 is an electrophoresis chart of RT-PCR amplification products of HF strain HA and NA gene, wherein a lane M is a DNA Marker; lane 1 is NA gene fragment; lane 2 is the HA gene fragment.
Detailed Description
Hereinafter, embodiments of the present invention will be described.
The term "vaccine composition" as used herein refers to a pharmaceutical composition containing the immunogenicity of avian influenza virus of subtype H9, which can induce, stimulate or enhance an immune response in chickens against avian influenza virus of subtype H9 only.
The term "immunizing amount" should be understood to mean an "immunologically effective amount," also known as an immunoprotective amount, or an amount effective to produce an immune response, that is an amount of antigen effective to induce an immune response in a recipient sufficient to prevent or ameliorate a sign or symptom of the disease, including adverse health effects or complications thereof. The immune response may be sufficient for diagnostic purposes or other tests, or may be suitable for preventing signs or symptoms of disease, including adverse health consequences or complications thereof caused by infection by a pathogen. Humoral immunity or cell-mediated immunity, or both, may be induced. The immune response of an animal to an immunogenic composition can be assessed indirectly, for example by measuring antibody titers, lymphocyte proliferation assays, or directly by monitoring signs or symptoms after challenge with a wild-type strain, while the protective immunity provided by a vaccine can be assessed by measuring, for example, clinical signs such as mortality, reduction in morbidity, temperature values, overall physiological condition and overall health and performance of the subject. The immune response may include, but is not limited to, induction of cellular and/or humoral immunity.
The term "H9 subtype avian influenza virus antigen" refers to any composition containing at least one form of H9 subtype avian influenza virus antigen which induces, stimulates or is capable of resisting an immune response against infection by H9 subtype avian influenza virus, including but not limited to inactivated, attenuated or subunit antigens.
The term "veterinarily acceptable carrier" refers to any other component in the vaccine composition of the present invention, except for the H9 subtype avian influenza virus antigen, which does not irritate the body and does not hinder the use of the biological activity and properties of the compound, or a carrier or diluent, preferably an adjuvant.
The term "adjuvant" may include aluminium gel adjuvants; saponins (saponin) such as Quil A, QS-21 (Cambridge Biotech Incorporation, cambridge MA), GPI-0100 (Galenica Pharmaceuticals Incorporation, birmingham AL); a water-in-oil emulsion; an oil-in-water emulsion; a water-in-oil-in-water emulsion; polymers of acrylic acid or methacrylic acid; a compound selected from copolymers of maleic anhydride and alkenyl (alk) derivatives. The term "emulsion" may be based in particular on light liquid paraffin oil (European Pharmacopea type); isoprenoid oils (isoprenoid oils) resulting from olefin oligomerization, such as squalane (squarane) or squalene oils (squarene oil), in particular isobutene or decene; linear alkyl-containing esters of acids or alcohols, more particularly vegetable oils, ethyl oleate, propylene glycol di- (caprylate/caprate), glycerol tri- (caprylate/caprate) or propylene glycol dioleate; esters of branched fatty acids or alcohols, especially isostearic acid esters. The oil is used in combination with an emulsifier to form an emulsion. The emulsifiers are preferably nonionic surfactants, in particular esters of sorbitan, mannide (e.g. anhydrous mannitol oleate), aliphatic diols (glycerol), polyglycerols (polyglycerol), propylene glycol and oleic acid, isostearic acid, ricinoleic acid or hydroxystearic acid, which are optionally ethoxylated, and also polyoxypropylene-polyoxyethylene block copolymers, in particular of the Pluronic product, in particular L121. See Hunter et al, the theory and practical application of adjuvants (ed.by DES Stewart-Tull, john Wiley and Sons, new York, 1995:51-94) and Todd et al, vaccine (1997, 15:564-570). For example, SPT emulsions described on page 147 and MF59 emulsions described on page 183 of Vaccine design, the Subunit and adiuvant approach (Plenum Press, 1995) written by Powell M and Newman M may be used. The term "polymer of acrylic acid or methacrylic acid" is preferably a crosslinked acrylic acid or methacrylic acid polymer, in particular with polyalkenyl ethers or polyols of sugars (sugam), known as carbomers (trade name Carbopol) (Phameuropa, 1996,8 (2)). Those skilled in the art are also referred to U.S. patent No. 2909462, which describes such acrylic polymers crosslinked with polyhydroxylated compounds having at least 3 hydroxyl groups, preferably no more than 8, wherein the hydrogen atoms of at least 3 hydroxyl groups are replaced by an unsaturated aliphatic radical having at least 2 carbon atoms (aliphatic radical). Preferred groups are those containing 2 to 4 carbon atoms, such as vinyl, allyl and other ethylenically unsaturated groups (ethylenically unsaturated group). The unsaturated group may itself contain other substituents, such as methyl. These products are sold in the name carbopol, (BF Goodrich, ohio, USA) which is particularly suitable. They are crosslinked with allyl sucrose or with allyl pentaerythritol (allyl pentaerythritol). Among these, carbopol 974P, 934P, and 971P may be mentioned, and carbopol 971P is most preferably used. The term "copolymers of maleic anhydride and alkenyl derivatives" also contemplates copolymers of maleic anhydride and ethylene EMA (Monsanto) which dissolve in water to give an acidic solution, which is neutralized, preferably to physiological pH, in order to give an adjuvant solution into which the immunogenic, immunogenic or vaccine composition itself can be incorporated. The term "adjuvant" also includes, but is not limited to, RIBI adjuvant system (Ribi Incorporation), block co-polymer (CytRx, atlanta GA), SAF-M (Chiron, emeryville Calif.), monophosphoryl lipid A (monophosphoryl lipid A), avridine lipid-amine adjuvant, E.coli heat labile enterotoxin (recombinant or otherwise), cholera toxin, IMS 1314, muramyl dipeptide, gel adjuvant, and the like. Preferably, the adjuvant comprises one or more of an aluminum Gel adjuvant, a saponin, a water-in-oil emulsion, an oil-in-water emulsion, a water-in-oil-in-water emulsion, a polymer of acrylic acid or methacrylic acid, a copolymer of maleic anhydride and an alkenyl (alkinyl) derivative, a RIBI adjuvant system, a Block co-polymer, SAF-M, a monophosphoryl lipid A, avridine lipid-amine adjuvant, an E.coli thermolabile enterotoxin, cholera toxin, IMS 1314, muramyl dipeptide, or Gel adjuvant.
The term "combination vaccine" refers to a vaccine prepared from a viral mixture of the avian influenza virus of subtype H9 of the present invention and at least one different virus. The term "composite vaccine" refers to vaccines prepared from the H9 subtype avian influenza virus and bacteria of the present invention. For example, the H9 subtype avian influenza virus of the present invention may be mixed or combined with newcastle disease virus, infectious bronchitis virus, egg drop syndrome virus, infectious bursal disease virus, adenovirus, reovirus and/or escherichia coli, avian parachicken bacillus, mycoplasma synoviae, mycoplasma gallisepticum.
The term "preventing and/or treating" when referring to an infection with an H9 subtype avian influenza virus refers to inhibiting replication of the H9 subtype avian influenza virus, inhibiting transmission of the H9 subtype avian influenza virus or preventing colonization of its host by the H9 subtype avian influenza virus, as well as alleviating the symptoms of a disease or disorder of the infection with the H9 subtype avian influenza virus. The treatment is considered to be therapeutic if the viral load is reduced, the condition is reduced, and/or the feed intake and/or growth is increased.
In order that the invention may be more readily understood, the invention will be described in more detail with reference to the following examples, which are intended to be illustrative only and are not intended to limit the scope of the invention, and the specific experimental methods not mentioned in the following examples are generally carried out in accordance with conventional experimental methods.
Example 1 isolation and identification of viruses
The low pathogenicity H9N2 subtype avian influenza virus is spread in a large scale in China, in recent years, chickens immunized with the H9N2 subtype avian influenza vaccine still infect the H9N2 subtype avian influenza in a plurality of areas, and the virus is suggested to possibly have a new epidemic trend through antigen drift or antigen transformation evolution, so that the virus finally causes immune failure, and a large amount of epidemiological investigation and research is carried out aiming at the variation condition of the virus, and finally, the H9 subtype avian influenza virus is successfully separated from a chicken farm morbidity group in the Hefei city of Anhui province.
1.1 isolation of viruses
Selecting a disease-causing chicken with pathological changes such as cough, sneeze and the like, visible bronchi embolism, tracheal mucosa edema and serous exudates and the like in a sectioning examination, taking internal organs (liver, kidney, spleen and the like) of the disease-causing chicken, adding PBS (0.01 mol/L, pH value of 7.0-7.4, penicillin 10000U/ml and streptomycin 2000U/ml) containing double antibodies, grinding, centrifuging for 15 minutes at 4 ℃ after grinding for 3000r/min, removing upper fat, sucking middle liquid, filtering by a 0.22 mu m filter, inoculating SPF chick embryo of 10 days old by using an allantoic cavity, incubating for 120 hours at 37 ℃, discarding dead chick embryo within 24 hours, harvesting dead chick embryo within 24-120 hours and allantoic fluid of survival chick embryo within 120 hours.
1.2 identification of viruses
1.2.1 Hemagglutination Assay (HA)
Chicken erythrocytes with 1% concentration were prepared, and the collected allantoic fluid was used as a test sample, and the HA titer of the allantoic fluid was detected by a Hemagglutination Assay (HA), which was set as a PBS negative control. HA titers greater than 1:16 were judged positive. The results showed that the aggregation titer of chick embryo allantoic fluid on 1% chick erythrocytes was 1:256.
1.2.2 hemagglutination inhibition assay (HI)
Based on the results of the HA assay, 4 working units of virus were formulated with PBS (pH 7.0-7.2). And (3) taking 4 units of virus as antigens to be detected, carrying out a hemagglutination inhibition test (HI) with standard chicken newcastle disease virus hemagglutination inhibition test positive serum, chicken egg drop syndrome virus hemagglutination inhibition test positive serum, avian influenza virus H9 subtype, H5 subtype and H7 subtype hemagglutination inhibition test positive serum, and identifying the type of the virus. The result shows that the isolated strain only has cross reaction with positive serum of an avian influenza virus H9 subtype hemagglutination inhibition test and has no cross reaction with other positive serum, which indicates that the allantoic fluid only contains H9 subtype avian influenza virus and has no newcastle disease virus, chicken egg drop syndrome virus, H5 subtype and H7 subtype avian influenza virus.
1.3 purification of viruses
Serial dilution of chick embryo allantoic fluid obtained in step 1.1 with 10 times sterile physiological saline to 10 -7 ～10 -10 The dilution, the allantoic fluid of each dilution is inoculated with SPF chick embryo of 9-11 days old through allantoic cavity, 0.1 ml/piece, chick embryo culture and allantoic fluid harvest are carried out according to the step 1.1. Detecting whether the allantoic fluid contains H9 subtype AIV or not through HA and HI experiments, inoculating SPF chick embryo with the allantoic fluid with the highest dilution, obtaining toxic allantoic fluid, continuously carrying out passage purification for 2 times according to the method, finally obtaining purified virus named HF strain E1 generation, and determining HA titer to be 1:512.
1.4 determination of subtype of viral isolates
1.4.1 design and Synthesis of primers
RT-PCR primers for amplifying the HA and NA genes were designed and synthesized with reference to AIV sequence data published on Genbank, and specific sequences are as follows.
HA-F：5’-AGCRAAAGCAGGGGAATTTCACAAC-3’
HA-R：5’-AGTAGAAACAAGGGTGTTTTTGCCAA-3’
NA-F：5’-AGCRAAAGCAGGAGTAAAAATGAAT-3’
NA-R：5’-AGTAGAAACAAGGAGTTTTTTCTAAAA-3’
1.4.2 extraction of purified viral RNA
The chick embryo allantoic fluid containing the purified virus is used as a material for extracting RNA, and the specification of a virus RNA extraction kit is referred to extract RNA of the purified virus.
RT-PCR of 1.4.3HA and NA Gene fragments
Preparing 50 μl RT-PCR reaction system by using the full gold One-Step RT-PCR Supermix kit and RNA extracted in Step 1.4.2 as raw material: mu.l of 2 XReation Mix, 1. Mu.l of Enzyme Mix, 1. Mu.l of each of primers NA-F and NA-R (or HA-F and HA-R), 18. Mu.l of RNase-free water, 4. Mu.l of RNA extracted in step 1.4.2. Reaction conditions: pre-denaturation at 45℃for 20 min, 94℃for 4 min; 30 cycles: 94℃for 30 seconds, 52℃for 30 seconds, 72℃for 100 seconds; finally, the sample is extended for 10 minutes at 72 ℃, and the result shows that the NA gene fragment with the size of 1400bp and the HA gene fragment with the size of 1600bp are successfully amplified, and the electrophoresis result is shown in figure 1.
1.4.4 sequencing and analysis
The RT-PCR products of step 1.4.3 were sent to sequencing company for nucleotide sequence determination, and based on the sequencing results, the Open Reading Frames (ORFs) of the HA and NA genes were aligned using the online alignment tool BLAST. The results show that the nucleotide homology of the HF strain and HA and NA genes of a plurality of H9N2 subtype avian influenza virus strains separated in 2013-2015 in China can be up to 99%, which indicates that the HF strain separated in the research is H9N2 subtype avian influenza virus and can represent the epidemic trend of H9 subtype avian influenza.
EXAMPLE 2 determination of chick embryo and chick pathogenicity
2.1 determination of the infection by half an embryo (EID 50)
Serial dilution of the separated virus with sterilized normal saline 10 times to obtain 10 -1 ～10 -9 Inoculating 10-11 day-old SPF chick embryo, 0.1 ml/piece of the diluted virus liquid into allantoic cavity, inoculating 5 chick embryos, discarding 24-hour dead chick embryos, freezing the dead chick embryos at 120 hours and 4 ℃ overnight after inoculation, determining the HA titer of the chick embryos, judging positive when the HA titer is greater than 1:16, and calculating the virus EID by referring to a Reed-Muench method 50 Is 10 9.17 /0.1ml。
2.2 virulence against SPF chick embryos
Diluting the HF strain E1 virus solution with sterilized normal saline 10 5 10 SPF chick embryos of 10 days old were inoculated with 0.1ml per embryo by multiple times via allantoic cavity. The death of 9 chick embryos and the survival of 1 chick embryo 24-96 hours after inoculation are observed. The dead chick embryo body has bleeding, edema and other lesions.
2.3 virulence against SPF chickens
To determine the intravenous inoculation pathogenicity index (IVPI), 10 SPF chickens of 6 weeks old were intravenously injected after 10-fold dilution of the HF strain E1 generation virus solution, each 0.1ml was observed for 10 days after challenge, no abnormal reaction was seen, the IVPI index was calculated to be 0, and the strain was determined to be a low pathogenic strain. At the same time, on day 5 after the challenge, collecting all the cotton swabs of the throat and cloaca of the challenge chicken, placing the cotton swabs in sterile PBS containing antibiotics (containing 10000U/ml penicillin and 8000U/ml streptomycin), mixing the two cotton swabs of the same chicken in equal quantity to be used as one sample, inoculating the sample into SPF chick embryo of 10-11 days old to separate viruses through allantoic cavities, and obtaining the result that 10/10 virus separation is positive.
EXAMPLE 3 viral antigenicity analysis
3.1 Preparation of H9 single factor serum
10H 9N2 subtype AIV isolated strains and HF strains related to the invention are respectively prepared into oil emulsion inactivated vaccine. The vaccine is respectively immunized on SPF chickens of 21 days old, 3 vaccine are immunized on each vaccine, secondary immunization is carried out 14 days after the first immunization, 0.3ml vaccine is immunized on leg muscle each time, the chickens are fed in a positive pressure isolator for poultry, sterile blood collection is carried out 21 days after the secondary immunization, serum is separated to prepare single factor positive serum, the single factor positive serum is packaged into small tubes, and the small tubes are preserved at the temperature of minus 20 ℃.
3.2 determination of antigen Cross-reactivity between different strains
The antigen correlation between the H9N2 viruses was determined by preparing 4 units of antigen from 11H 9N2 subtype AIVs of 3.1 vaccine, then performing a blood coagulation inhibition test (HI) by crossing each other with the single-factor serum prepared in 3.1, repeating the test three times, and taking the average value of the three tests for analysis of the antigen difference coefficient R value. R-value calculation methods are described in the second edition of animal virology (Yan Zhen et al, 1997).
r1=hemagglutination inhibition titre of nail serum against b antigen ≡hemagglutination inhibition titre of nail serum against a antigen
r2=hemagglutination inhibition titre of b serum against a antigen ≡hemagglutination inhibition titre of b serum against b antigen
Judgment standard: r=1, indicating that both strains are antigenically identical; r is more than or equal to 0.67 and less than 1, which shows that the two strains have no obvious difference; r is more than or equal to 0.5 and less than 0.67, which shows that the antigenicity of the two strains has smaller difference; r is less than 0.5, which shows that the antigen difference of the two strains is obvious; the smaller the R value, the greater the antigenic difference between the two strains. The antigen cross-reaction results were as follows:
the results of the HI cross-test between different H9N2 subtype AIV strains and the results of strain antigen correlation analysis are shown in tables 1 and 2, respectively. As shown in Table 2, the antigen correlation between the HF strain and the H9N2 virus separated after 14 years is more than 0.85, which shows that the virus has good antigen correlation with epidemic strains, and can be used as a candidate strain of H9N2 subtype avian influenza vaccine. And submitting for preservation.
TABLE 1 HI crossover test results between different H9N2 subtype AIV strains
TABLE 2 correlation of different H9N2 AIV strains antigen (R values)
Example 4 preparation of inactivated vaccine
4.1 preparation of virus liquid
Taking H9 subtype avian influenza virus HF strain E1 generation virus liquid, and diluting to 10 by using sterile normal saline -3 (0.1 ml of virus liquid is added into 0.9ml of sterile physiological saline, and diluted for 2 times in sequence after shaking and mixing), 10-day-old SPF chick embryos (self-hatching by using SPF hatching eggs purchased from biological technology Co., ltd. In Yinhe, beijing) are inoculated through the allantoic cavity, and each embryo is 0.1ml. Sealing the pinhole after inoculation, and placing at 36-37 ℃ for continuous incubation without turning over the eggs. Taking out until 96 hours, standing the air chamber upwards, and cooling for 12-24 hours at the temperature of 2-8 ℃. And (5) harvesting embryo liquid from the cooled chick embryo. The obtained virus liquid was sampled and the virus content was measured according to the method described in appendix 7 of the pharmacopoeia of the people's republic of China (2010 edition) and was 10 9.0 EID 50 /0.1ml。
4.2 antigen inactivation
The analytically pure formaldehyde solution was diluted 10 times with purified water and added to the virus solution of step 4.1 with shaking to a final concentration of 0.1% (V/V). After shaking and mixing, transferring into another sterile container, sealing, inactivating at 37 deg.C for 24 hr, shaking for 1 time at intervals of 4-6 hr.
4.3 emulsification
Mixing 94 parts by volume of white oil for injection and 806 parts by volume of span, adding 2 parts by mass of aluminum stearate, stirring until the mixture is transparent, and sterilizing under high pressure for cooling to obtain an oil phase.
Preparing the inactivated avian influenza virus liquid in the step 4.2 into 10 with sterile normal saline 8.5 EID 50 And (3) placing the antigen concentration of 0.1ml in a sterilization container, adding sterilized Tween-80 according to the volume of 4% (V/V) of the virus liquid, and fully shaking to completely dissolve the Tween-80 to obtain the water phase.
2 parts by volume of the oil phase is taken and placed in an oil phase tank, a motor is started to stir slowly, then 1 part by volume of the water phase is slowly added, the mixture is transferred into an emulsifying tank after the addition is completed, and then the mixture is emulsified for 40 minutes at 2800 revolutions per minute, namely 1% (W/V) sulfur Liu Gongna solution is added before the emulsification is terminated, so that the final concentration of the thimerosal sodium is 0.01% (W/V). The specific proportions are shown in Table 3.
Table 3 H9 subtype avian influenza virus inactivated vaccine formulation
| Component (A) | Vaccine 1 | Vaccine 2 | Vaccine 3 |
| HF strain antigen (EID) 50 /0.1ml) | 10 6.5 | 10 8.0 | 10 8.5 |
| White oil adjuvant (V/V%) | 66％ | 66％ | 66％ |
Example 5 immunoprotection test of HF Strain
5.1 serological methods
The inactivated vaccine prepared by the HF strain of the H9 subtype avian influenza virus of example 4 is injected subcutaneously into 0.3ml of 35 SPF chickens of 3 weeks of age, 10 chickens of each group, 3 groups, and the other 5 non-immunized chickens are used as a control. Each chicken was sampled 21 days after inoculation, serum was isolated and HI antibody titers were determined according to the current chinese beast pharmacopoeia. The geometric mean of the HI antibody titers of the immunized group was 1:630 (micro method), and the geometric mean of the HI antibody titers of the non-immunized control group was not higher than 1:2 (micro method), and the detection results are shown in Table 4.
TABLE 4 serum AI HI antibody titres detection results 21 days post-exemption
Note that: "/" indicates that this item is content free, and "GMT" indicates a geometric mean.
The results show that the HI antibody titers of the vaccine compositions of the invention are much higher than 6.0 logs 2 Vaccine standard.
5.2 immune toxicity counteracting method
Using 35 chickens of 5.1, each chicken was intravenously injected with avian influenza (H9 subtype) virus HF strain virus solution, 0.2 ml/chicken (containing 10) after 21 days of immunization 7.0 EID 50 ). Collecting throat and cloaca swab of each chicken on day 5 after virus attack, mixing throat and cloaca swab of the same chicken as 1 sample, inoculating 5 SPF chick embryos of 10 days old with allantoic cavity, each embryo being 0.2ml, incubatingThe embryo-by-embryo assay was performed for 120 hours to determine the HA titer of the chick embryo. The virus isolation positive can be judged as long as the HA titer of 1 chick embryo liquid in 5 chick embryos inoculated with each swab sample is not less than 1:16 (micro method). For a sample which is negative in virus separation, the judgment should be carried out after 1 generation of blind transmission. The results are detailed in Table 5.
TABLE 5 Virus isolation results after challenge
| Group of | Positive rate of viral isolation |
| Vaccine 1 | 0/10 |
| Vaccine 2 | 0/10 |
| Vaccine 3 | 0/10 |
| Control group | 5/5 |
The result shows that the vaccine composition has the virus separation rate of 0 through the efficacy test of an immune toxicity attack method, which proves that after the vaccine composition is immunized, chickens are not detoxified, the infection of H9 subtype avian influenza is completely resisted, and the vaccine composition has a good immune protection effect.
Example 6 immune toxicity-counteracting Cross-protection test of HF strain
Taking 75 SPF chickens of 3 weeks old, wherein 60 chickens are divided into 10 chickens/group 1-6 groups, and 1-3 groups are subjected to subcutaneous or leg muscle immunization, wherein 2,0.3 ml/person of inactivated vaccine prepared from the H9 subtype avian influenza virus HF strain in the embodiment 4 of the invention; 4-6 groups of commercial vaccine chicken newcastle disease and avian influenza (H9 subtype) bivalent inactivated vaccine (LaSota strain+WD strain) with 0.3 ml/feather of muscle inoculation according to the specification; the other 15 were divided into 5 pieces/group as control group. After 3 weeks of immunization, each chicken was collected separately, serum was isolated, HI antibody titer was measured, 1-3 groups were tested for HI antigen prepared from H9 subtype avian influenza virus HF strain, 4-6 groups were tested for HI antigen from avian influenza (H9 subtype) by halbinavidae biotechnology development company, the results showed that the immunized groups all reached higher antibody levels, and then epidemic strains a518-2015, a17089-2017, a182096-2018 were used to challenge each chicken, throat and cloaca swabs were collected, the throat and cloaca swabs of the same chicken were mixed and used as 1 sample, 10 day old SPF chick embryos were inoculated through the allanto cavity, 0.2ml each embryo was inoculated and observed for 120 hours, and the HA titer of chick embryo was measured embryo by embryo. The virus isolation positive can be judged as long as the HA titer of 1 chick embryo liquid in 5 chick embryos inoculated with each swab sample is not less than 1:16 (micro method). For a sample which is negative in virus separation, the judgment should be carried out after 1 generation of blind transmission. The results are detailed in Table 6.
TABLE 6 challenge test of avian influenza H9 subtype epidemic strains
Note that: "/" means that the item has no content
After a commercial vaccine chicken newcastle disease for preventing H9 subtype avian influenza and an avian influenza (H9 subtype) bivalent inactivated vaccine (LaSota strain+WD strain) are used for immunizing SPF chickens, H9 subtype avian influenza A518-2015, A17089-2017 and A182096-2018 strains separated in 2015-2018 are used for attacking, and virus elimination is detected after 5 days of attacking, so that the virus detection rate of the attacking chickens is above 30%, namely the early H9 subtype avian influenza commercial vaccine in the experiment can generate high-level antibodies after immunization, but cannot resist the attack of 2015-2018 epidemic strains. The inactivated vaccine of the invention can be completely protected.
EXAMPLE 7 preparation of newcastle disease antigen
Newcastle disease virus (gene VII type), N7a strain (Newcastle Disease Virus (genotype VII), strain N7 a) (collection in China center for type culture Collection, with a collection number of CCTCC NO: v20145, a preservation date of 2015, 10 month and 19 days, a preservation address of university of Wuhan, china, published in China patent application CN 107281479A), properly diluted with sterilized physiological saline (10) -4 Or 10 -5 ) Inoculating 10-11 days old susceptible chick embryo with 0.1ml each embryo, and incubating at 37 deg.c. Selecting dead and alive chick embryo 48-120 hours after inoculation, harvesting allantoic fluid, and determining virus content to be 10 8.0 EID 50 0.1ml. Adding formaldehyde solution (v/v) with the final concentration of 0.1%, inactivating at 37 ℃ while stirring for 16h every 4-6 h, and completely inactivating for later use.
Example 8 preparation of antigen for infectious bronchitis in chickens
The infectious bronchitis virus M41 strain (purchased from China veterinary drug administration) was properly diluted with sterilized physiological saline (10) -2 Or 10 -3 ) Inoculating susceptible chick embryo of 10-11 days old, each embryo is 0.1ml, and incubating at 36-37 deg.C. Selecting dead and alive chick embryo 24-48 hours after inoculation, harvesting allantoic fluid, and determining virus content to be 10 6.0 EID 50 0.1ml. Adding formaldehyde solution (v/v) with the final concentration of 0.1%, placing at 37 ℃ for inactivation, stirring for 16h every 4-6 h during the inactivation, and completely inactivating for later use.
EXAMPLE 9 preparation of avian egg drop syndrome antigen
The egg-reducing syndrome virus HX Strain (Egg Drop syndrome Virus, stress HX, preserved in China Center for Type Culture Collection (CCTCC) NO: V201942, the preservation date is 2019, 06 months and 19 days, the preservation address is Wuhan and Wuhan university, china), diluted by proportion with sterile physiological saline, allantoic cavity inoculated with 10-day-old susceptible duck embryo, each embryo is 0.1ml, the inoculated duck embryo is placed at 36-37 ℃ for continuous incubation, dead duck embryo is discarded before 24 hours, eggs are taken 1 time every 6-8 hours, the dead duck embryo is taken out at any time until 120 hours, all duck embryos are taken out, the air chamber stands upwards, and the duck embryo is placed at 2-8 ℃ for cooling for 12-24 hours; then the allantoic fluid of the duck embryo is obtained aseptically, and the virus content is measured to be 10 8.5 EID 50 0.1ml. AddingAdding formaldehyde solution (v/v) with the final concentration of 0.2%, inactivating at 37 ℃ while stirring for 16h every 4-6 h, and completely inactivating for later use.
EXAMPLE 10 preparation of bursa Fabricius antigen
Preparation of VP2 cDNA
IBDV RNA was extracted from SPF chicken bursa of Fabricius infected with the virulent capital strain of infectious bursal disease virus according to the viral RNA extraction kit procedure and reverse transcribed with random primers. Oligonucleotide primers were synthesized based on the sequences of the conserved regions at the 5 'and 3' ends of the VP2 gene, the sequences of the synthesized oligonucleotide primers are shown in Table 7, and PCR amplification was performed, and recovered using an agarose gel recovery kit, and stored at-20 ℃.
TABLE 7 bursal disease virus VP2 gene amplification primers
Construction of pCold III-VP 2/E.Coli BL21 (DE 3) Strain
Taking the prepared VP2 cDNA, carrying out double enzyme digestion, and connecting the digested fragments to a pCold III carrier; the ligation product was directly transformed into E.coli BL21 (DE 3) and spread on solid medium containing 100. Mu.g ampicillin LB for overnight culture, and the grown colony was pCold III-VP 2/E.Coli BL21 (DE 3) strain.
3. Preparation of chicken infectious bursal disease virus VP2 protein
The culture tank is used for aeration culture, and 70% of culture medium and peanut oil defoamer are filled according to volume. Inoculating seed solution of pCold III-VP 2/E.Coli BL21 (DE 3) strain according to 2% -4% of the culture medium amount after sterilization, culturing at 37 ℃, adding 0.2mol/L alpha-lactose when the OD600 value of the bacterial solution reaches 0.6-1.0, enabling the final concentration to reach 0.02mol/L, and continuing culturing for 5-8 hours.
After the culture, the cells were collected by centrifugation, resuspended, sonicated, and the supernatant was collected by centrifugation. After precipitation with ammonium sulfate, the VP2 protein solution was collected.
EXAMPLE 11 preparation of avian adenovirus antigen
Taking fowl adenovirus FAV-HN strain (preserved in China center for type culture collection (CCTCC NO: V201609, date of preservation is 2016, 02 and 29 days), and preserved at university of Wuhan and Wuhan, disclosed in Chinese patent application CN 107523556A), properly diluting with sterilized normal saline (10) -4 Or 10 -5 ) Inoculating 5-7 days old susceptible chick embryo with 0.1ml each embryo, and incubating at 37 deg.c. Selecting dead and alive chick embryo 24-144 hours after inoculation, harvesting allantoic fluid, and determining the virus content to be 10 8.5 EID 50 0.1ml. Adding formaldehyde solution (v/v) with the final concentration of 0.1%, inactivating at 37 ℃ for 24 hours after stirring every 4-6 hours.
Example 12 preparation of an avian influenza subtype H9 Virus combination vaccine
The H9 subtype avian influenza virus antigen prepared in example 4 was mixed with the newcastle disease antigen prepared in example 7, the infectious bronchitis antigen prepared in example 8, the egg drop syndrome antigen prepared in example 9, the infectious bursal antigen prepared in example 10, and the adenovirus antigen prepared in example 11, respectively, in proportion, and then added into white oil adjuvant, and simultaneously, the motor was started for 17500r/min to stir for 5min, and 1% merthiolate solution was added before stopping stirring to make the final concentration 0.01%. The specific proportions are shown in tables 8, 9, 10 and 11.
Table 8 H9 subtype avian influenza virus bivalent vaccine formulation
| Component (A) | Vaccine 4 | Vaccine 5 | Vaccine 6 | Vaccine 7 | Vaccine 8 |
| HF strain antigen (EID) 50 /0.1ml) | 10 6.5 | 10 7.0 | 10 7.5 | 10 8.0 | 10 8.5 |
| N7a Strain antigen (EID 50 /0.1ml) | 10 8.0 | — | — | — | — |
| M41 Strain antigen (EID) 50 /0.1ml) | — | 10 6.0 | — | — | — |
| HX strain antigen (EID) 50 /0.1ml) | — | — | 10 7.0 | — | — |
| VP2 protein (AGP potency) | — | — | — | 1：16 | — |
| FAV-HN strain antigen (EID 50 /0.1ml) | — | — | — | — | 10 6.5 |
| White oil adjuvant (V/V%) | 66％ | 66％ | 66％ | 66％ | 66％ |
Table 9 H9 subtype avian influenza virus triple vaccine formulation
| Component (A) | Vaccine 9 | Vaccine 10 | Vaccine 11 | Vaccine 12 |
| HF strain antigen (EID) 50 /0.1ml) | 10 6.5 | 10 7.0 | 10 7.5 | 10 8.0 |
| N7a Strain antigen (EID 50 /0.1ml) | 10 8.0 | 10 8.0 | 10 8.0 | 10 8.0 |
| M41 Strain antigen (EID) 50 /0.1ml) | 10 6.0 | — | — | — |
| HX strain antigen (EID) 50 /0.1ml) | — | 10 7.0 | — | — |
| VP2 protein (AGP potency) | — | — | 1：16 | — |
| FAV-HN strain antigen (EID 50 /0.1ml) | — | — | — | 10 6.5 |
| White oil adjuvant (V/V%) | 66％ | 66％ | 66％ | 66％ |
Table 10 H9 subtype avian influenza virus tetrad vaccine ratio
Table 11 H9 subtype avian influenza virus quintuplet vaccine mixture ratio
| Component (A) | Vaccine 19 | Vaccine 20 |
| HF strain antigen (EID) 50 /0.1ml) | 10 8.0 | 10 8.0 |
| N7a Strain antigen (EID 50 /0.1ml) | 10 8.0 | 10 8.0 |
| M41 Strain antigen (EID) 50 /0.1ml) | 10 6.0 | 10 6.0 |
| HX strain antigen (EID) 50 /0.1ml) | 10 7.0 | — |
| VP2 protein (AGP potency) | — | 1：16 |
| FAV-HN strain antigen (EID 50 /0.1ml) | 10 6.5 | 10 6.5 |
| White oil adjuvant (V/V%) | 66％ | 66％ |
Example 13 immunogenicity test of avian influenza subtype H9 virus combination vaccine
1. Partial immunogenicity test of avian influenza
180 SPF chickens of 21 days old are divided into 18 groups, 10 chickens are respectively taken from 10 groups to 26 groups, and 4 to 20 ml/1 of vaccine prepared in the immunization example 12 are respectively injected into the neck subcutaneous injection; group 27 was subcutaneously injected with 0.3ml of physiological saline as a blank. All test chickens were kept separate, and on day 21 post immunization, 10 th to 26 th immunized chickens, along with 27 th control chickens, were collected and serum was isolated. Detecting the titer of the HI antibody of the H9 subtype avian influenza and simultaneously using HF Strain virus solutions were challenged by intravenous injection, each 0.2ml (containing 10 7.0 EID 50 ). Collecting cloacal swabs 5 days after virus attack, inoculating 5 SPF chick embryos of 10-11 days old into allantoic cavities after treatment, incubating and observing for 5 days, and determining the erythrocyte agglutination value of chick embryo liquid no matter dead embryo or living embryo, wherein the agglutination value of 1 chick embryo liquid in 5 chick embryos inoculated by each swab sample is not lower than 1:16 (micro method), and judging that the virus separation is positive. Samples negative for virus isolation should be determined after blind transmission. The immune group should be negative for at least 9 chicken viruses to isolate; the control group should be positive for at least 4 chicken viruses. The results are shown in Table 12.
Table 12 results of partial immunogenicity test of avian influenza subtype H9 virus combination vaccine
Note that: HI antibodies were determined as the geometric mean of immunized chicken antibodies.
The results show that the vaccines 4-20 can generate higher avian influenza antibodies 21 days after immunization, and compared with a control, the immune group can completely protect against virulent attack. The H9 subtype avian influenza virus liquid provided by the invention is shown to be used as an oil emulsion joint vaccine prepared by antigens, so that complete protection can be provided for chicken flocks.
2. Newcastle disease partial immunogenicity assay
Taking 140 SPF chickens of 21 days old, dividing the SPF chickens into 14 groups, 10 each, 28 th to 40 th groups respectively carrying out neck subcutaneous injection on vaccine 4, 9 th to 20 th vaccines and 20 mu l/each prepared in the immunization example 12; group 41 was subcutaneously injected with 20 μl of saline as a blank. All test chickens were kept separate, and on day 21 post immunization, 28 th to 40 th immunized chickens, along with 41 st control chickens, were collected and serum was isolated. Detecting the HI antibody of the newcastle disease virus, simultaneously using the strong HN1101 strain virus liquid of the newcastle disease virus to attack through intramuscular injection, observing for 14 days, and recording the number of morbidity, mortality and protection. The results are shown in Table 13.
TABLE 13 results of partial immunogenicity test of avian influenza subtype H9 virus combination vaccine newcastle disease
Note that: HI antibodies were determined as the geometric mean of immunized chicken antibodies.
The results show that the immune groups of the vaccine 4 and the vaccine 9-20 can generate higher newcastle disease antibodies 21 days after immunization, and compared with the control, the immune groups can completely protect against virulent attack. The N7a strain newcastle disease virus liquid provided by the invention is used as an oil emulsion joint vaccine prepared by antigens to provide complete protection for chicken flocks.
3. Partial immunogenicity test for infectious bronchitis
80 SPF chickens of 21 days old are divided into 8 groups of 10 chickens, and each eye of 42 th group to 48 th group is inoculated with 1 feather fraction (0.05 ml) of chicken infectious bronchitis live vaccine (H120 strain) by nasal drip. 21 days after inoculation, along with group 49 control chickens, blood was collected and serum was isolated. Meanwhile, each of 42 to 48 groups was immunized with 0.3 ml/dose of vaccine 5, vaccine 9, vaccine 13, vaccine 14, vaccine 15, vaccine 19, vaccine 20, prepared in example 12, by subcutaneous injection in the neck. 28 days after inoculation, blood is taken and serum is separated from the control chickens of the 49 th group respectively; the serum collected twice on 21 days after the first immunization of the live vaccine and 28 days after the first immunization of the immunized chicken of the 42 th to 48 th groups (the serum collected at the same time as the control chicken of the 49 th group) is used for measuring the HI antibody titer. The geometric mean of the immune group di-immune serum HI antibody titers is not lower than 4 times that of the primary immune serum HI antibody titers, and the geometric mean of the non-immune control group serum HI antibody titers is not higher than 1:8 (micro method). Simultaneously, the infectious bronchitis M41 virulent virus is used for attacking toxin 10 per feather drop nose 3.0 EID50, challenge experiment. The results are shown in Table 14.
TABLE 14 results of immunogenicity test of chicken transfer part of H9 subtype avian influenza virus combined vaccine
The results show that the geometric average value of the HI antibody titers of the second immune serum of the vaccine 5, the vaccine 9, the vaccine 13, the vaccine 14, the vaccine 15, the vaccine 19 and the vaccine 20 is not lower than 4 times of the geometric average value of the HI antibody titers of the first immune serum, and viruses are not separated from the air pipes of all immunized chickens after the virus attack, so that the attack of strong viruses can be completely protected. The oil emulsion combined vaccine prepared by using the chicken infectious bronchitis virus liquid provided by the invention as an antigen can provide complete protection for chicken flocks.
4. Partial immunogenicity test of avian egg drop syndrome
Taking 70 SPF chickens of 21 days old, dividing the SPF chickens into 7 groups, 10 each, and respectively carrying out neck subcutaneous injection on vaccine 6, vaccine 10, vaccine 13, vaccine 16, vaccine 17, vaccine 19 and 0.3 ml/dose prepared in immunization example 12 from 50 th to 55 th groups; group 56 was subcutaneously injected with 0.3ml of physiological saline as a blank. All test chickens are kept separately, blood is collected from 50 th group to 56 th group 21 days after immunization, the HI antibody titer of the egg drop syndrome of the poultry is measured, and the geometric average titer of the HI antibody of the immunized chickens is more than or equal to 7log 2 The HI antibody titer of the control chicken is less than or equal to 2log 2 . The results are shown in Table 15.
TABLE 15 results of immunogenicity test of egg-reducing portion of H9 subtype avian influenza virus combination vaccine
Note that: HI antibodies were determined as the geometric mean of immunized chicken antibodies.
The results show that the vaccine 6, the vaccine 10, the vaccine 13, the vaccine 16, the vaccine 17 and the vaccine 19 can generate higher egg drop syndrome antibodies 21 days after immunization, and can effectively protect the egg drop syndrome of the chicken flocks. The egg drop syndrome virus liquid provided by the invention is used as an oil emulsion combined vaccine prepared by antigens, so that complete protection can be provided for chicken flocks.
5. Bursa fabricius partial immunogenicity test
Taking 70 SPF chickens of 21 days old, dividing the SPF chickens into 7 groups, 10 chickens in each group, and respectively carrying out neck subcutaneous injection on vaccine 7, vaccine 11, vaccine 14, vaccine 16, vaccine 18, vaccine 20 and 0.3 ml/chicken which are prepared in immunization example 12 in groups 57-62; group 63 was subcutaneously injected with 0.3ml of saline as a blank. All test chickens were kept separately and raised, and on 21 days after immunization, groups 57 to 63 were inoculated with 100-fold dilutions of infectious bursal disease virulent BC6-85 (CVCC AV7 strain) from 0.1ml of virus liquid (real toxin content: 100 BID) of the strain of Chinese veterinary drug administration by each eye-drop route. After the toxicity attack, the clinical manifestations of chickens are observed every day, the numbers of the sick and dead chickens are recorded, the surviving chickens are killed for 72-96 hours, the chickens are dissected one by one, and the lesions such as bursa of Fabricius are observed. At least 8 immunized chickens should be normal without bursal disease; the control chicken should have at least 4 chicken diseases, and have obvious bursal disease (such as pectoral muscle or leg muscle strip hemorrhage, bursal enlargement or atrophy, yellowing, and jelly-like secretion). The results are shown in Table 16.
Table 16 results of the combined vaccine bursa of Fabricius partial immunogenicity test for subtype H9 avian influenza virus
The results show that vaccine 7, vaccine 11, vaccine 14, vaccine 16, vaccine 18, vaccine 20 can completely protect against virulent challenge of infectious bursal disease in chickens 21 days after immunization. The oil emulsion joint vaccine prepared by using the bursa fabricius VP2 protein provided by the invention as an antigen can provide complete protection for chicken flocks.
6. Avian adenovirus partial immunogenicity assay
Taking 80 SPF chickens of 21 days old, dividing the SPF chickens into 8 groups, 10 chickens in each group, and respectively carrying out neck subcutaneous injection on vaccine 8, vaccine 12, vaccine 15, vaccine 17-vaccine 20 prepared in immunization example 12 in 64 th to 70 th groups, wherein the volume of each group is 0.3 ml/one; group 71 was subcutaneously injected with 0.3ml of physiological saline as a blank. All test chickens were kept separately, challenged with FAV-HN strain virus solution by intramuscular injection 21 days after immunization, observed for 14 days, and the number of morbidity, mortality and protection was recorded. The results are shown in Table 17.
Table 17 results of avian influenza virus subtype H9 Combined vaccine avian gland part immunogenicity test
The results show that the immune groups of the vaccine 8, the vaccine 12, the vaccine 15 and the vaccine 17-20 can generate better immune protection 21 days after immunization. The oil emulsion combined vaccine prepared by using the avian adenovirus liquid provided by the invention as an antigen can provide complete protection for chicken flocks.
Proved by the invention, the H9 subtype avian influenza virus combined vaccine provided by the invention can resist the invasion of related pathogens, shows good immunogenicity, and can effectively control the epidemic of H9 subtype avian influenza virus related diseases in China.
EXAMPLE 14 preparation of an egg-drop syndrome Virus vaccine composition
The antigen of egg drop syndrome prepared in example 9 was added to white oil adjuvant while the motor was started, and stirred for 5min at 17500r/min, and 1% merthiolate solution was added to a final concentration of 0.01% before stopping stirring. The specific proportions are shown in Table 18.
Table 18 egg-reducing syndrome virus vaccine formulation
| Component (A) | Vaccine 21 | Vaccine 22 | Vaccine 23 |
| HX strain antigen (EID) 50 /0.1ml) | 10 7.0 | 10 7.5 | 10 8.0 |
| White oil adjuvant (V/V%) | 66％ | 66％ | 66％ |
EXAMPLE 15 immunogenicity test of egg drop syndrome Virus vaccine composition
Taking 40 SPF chickens of 21 days old, dividing the SPF chickens into 4 groups, 10 each, and performing neck subcutaneous injection on vaccine 21, vaccine 22 and vaccine 23 prepared in immunization example 14 from 72 th to 74 th, wherein each group is 0.3 ml/one; group 75 was subcutaneously injected with 0.3ml of physiological saline as a blank. All test chickens are kept separately, blood is collected from 72 th group to 75 th group 21 days after immunization, the HI antibody titer of the egg drop syndrome is measured, and the geometric average titer of the HI antibody of the immunized chickens is more than or equal to 7log 2 The HI antibody titer of the control chicken is less than or equal to 2log 2 . The results are shown in Table 19.
TABLE 19 results of immunogenicity testing of egg drop syndrome virus vaccine compositions
Note that: HI antibodies were determined as the geometric mean of immunized chicken antibodies.
The results show that the vaccine 21, the vaccine 22 and the vaccine 23 can generate higher egg drop syndrome antibodies after 21 days of immunization, and can effectively protect the egg drop syndrome of the chicken flocks. The oil emulsion vaccine prepared by using the egg drop syndrome virus liquid provided by the invention as an antigen can provide complete protection for chicken flocks.
The present invention is not limited to the above-mentioned embodiments, but is capable of modification and variation in all embodiments without departing from the spirit and scope of the present invention.
Claims (6)
1. A vaccine composition, which is characterized by comprising an immune amount of inactivated antigen of an HX strain of avian egg drop syndrome virus, inactivated antigen of an H9 subtype avian influenza HF strain and a pharmaceutically acceptable carrier, wherein the preservation number of the HX strain of avian egg drop syndrome virus is CCTCC NO: v201942, the collection number of the H9 subtype avian influenza HF strain is CCTCC NO: v201941.
2. The vaccine composition of claim 1, wherein the inactivated antigen content of the HX strain of avian egg drop syndrome virus is 10 7.0 ~10 8.0 EID 50 /0.1ml。
3. The vaccine composition of claim 1, wherein the pharmaceutically acceptable carrier is an adjuvant.
4. A vaccine composition according to claim 3, characterized in that the adjuvant is a white oil adjuvant, which is used for the preparation of a water-in-oil emulsion.
5. A vaccine composition according to claim 3, wherein the adjuvant is present in a concentration range of 66% V/V.
6. Use of the vaccine composition of any one of claims 1 to 5 in the manufacture of a medicament for preventing egg drop syndrome in poultry.
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