Attenuating method of duck short beak and dwarf syndrome virus and application of attenuating method

Abstract

The invention discloses a duck short beak and dwarf syndrome (SBDSV) virus attenuating method and application thereof. The attenuating method comprises the following steps: (1) sequentially performing acidic condition domestication and alkaline condition domestication on duck short beak and dwarf syndrome virus virulent strains; (2) carrying out single colony cloning on the strains treated in the step (1), and screening out strains with low toxicity and good immunogenicity through toxicity and immunogenicity determination; and (3) repeating the steps (1) and (2) for a plurality of times to obtain the duck short beak and dwarf syndrome virus attenuated strain with good safety and high immune efficacy. The duck short beak and dwarf syndrome virus strain is treated in acidic and alkaline environments to obtain a low virulent strain with low virulence and strong immunogenicity, LMH cells are inoculated for suspension culture, and the titer of the harvested virus liquid can reach 109.0 TCID50 / mL. The vaccine is prepared from the vaccine, the vaccine is good in safety, and an immune group can generate 100% immune protection effect.

Description

Technical Field

[0001] The present invention relates to veterinary vaccines, and in particular, to a method for attenuating duck short beak and dwarfism syndrome virus and its application. Background Art

[0002] Duck short beak and dwarfism syndrome virus is a new type of goose parvovirus, which mainly infects muscovy ducks, mule ducks, mallards, Cherry Valley Pekin ducks, etc. It is characterized by retarded development of ducklings, shortening of the upper and lower beaks, swelling of the tongue protruding outside, and easy fracture of the tibia and wing bones in the later stage of infection. Since the beginning of 2015, infected cases have been successively found in Anhui, Jiangsu, Shandong and other places in China. The tongues of diseased ducks protrude outside the beak and cannot freely eat and drink water, resulting in slow growth. Moreover, lame ducks with broken legs and wings are likely to appear during slaughter. This disease has become one of the important diseases endangering the breeding of meat ducks.

[0003] In order to obtain lasting immune protection, inactivated vaccines and subunit vaccines need to be immunized multiple times, which increases the burden on immunized animals and may lead to a decline in the immune effect. Inactivated vaccines do not multiply in animals, have poor antigenicity, induce low immune responses and protection effects, and usually need to rely on adjuvants to enhance the immune effect. Therefore, a large inoculation dose is required during use. The components and processes of adjuvants will affect the immunogenicity of inactivated vaccines, and the residues of inactivators in adjuvants and preparations will also cause side effects. Subunit vaccines also have poor immunogenicity and single antigen. Their cross-immune protection against different strains of the same serotype is uncertain, and they require the participation of immune adjuvants. Moreover, the body can only produce humoral immunity after immunization. Live attenuated vaccines have extremely strong immunogenicity, can stimulate the body to produce both humoral immunity and cellular immunity, and can protect the body 14 days after immunization.

[0004] Usually, duck short beak and dwarfism syndrome virus is amplified and propagated on duck embryo fibroblasts, and it takes more than 90 passages to obtain an attenuated strain. Duck embryo fibroblasts are primary cells, which are cumbersome to prepare, easily contaminated with exogenous viruses, and have a low virus titer. Summary of the Invention

[0005] Aiming at the problems of cumbersome method for attenuating duck short beak and dwarfism syndrome virus and low virus titer, the present invention provides a method for attenuating duck short beak and dwarfism syndrome virus and its application. Acidic and alkaline environment treatments are adopted for attenuating the virus strain, which is simple and convenient, and the effect is better than the traditional method. Suspension cell culture is used for virus amplification, and a virus solution with a higher titer and more yield can be obtained.

[0006] To achieve the above object, on the one hand, the present invention provides a method for attenuating duck short beak and dwarfism syndrome virus, which comprises the following steps: (1), subjecting the strong strain of duck short beak and dwarfism syndrome virus to acid condition domestication and alkaline condition domestication in sequence; (2) Perform single colony cloning on the virus strain processed in step (1), and screen out the virus strain with low virulence and good immunogenicity through virulence and immunogenicity determination; (3) Repeat steps (1) and (2) several times to obtain a weakly virulent duck short beak and dwarfism syndrome virus strain with good safety and high immune efficacy.

[0007] Standards for good safety and high immune efficacy: After inoculating ducklings with a 10-fold dose and observing for 14 days, if the ducklings are in good spirits and have no adverse reactions, it proves good safety. When inoculating 1 dose of ducklings, the geometric mean titer of serum neutralizing antibodies in ducklings 21 days after immunization should not be lower than 1:16, and it can resist the attack of virulent virus.

[0008] Specifically, in step (1), the screening method for the highly virulent duck short beak and dwarfism syndrome virus strain is as follows: Diseased ducks with typical duck short beak and dwarfism syndrome that broke out in a certain Cherry Valley duck farm in Jining City, Shandong Province were aseptically dissected, and their spleen, kidney and other diseased materials were used to isolate the virus with healthy non-immune Cherry Valley duck embryos; By comparing and analyzing its DNA sequence and amino acid sequence with the classical GPV and the published duck short beak and dwarfism syndrome sequences, a typical duck short beak and dwarfism syndrome virus was successfully isolated.

[0009] Specifically, in step (1), the acid condition domestication is to place it at pH 4.5 - 5.5 for 4 - 5 hours; and / or The alkaline condition domestication is to place it at pH 9.0 - 10.0 for 4 - 5 hours.

[0010] Preferably, in step (3), steps (1) and (2) are repeated 4 - 6 times.

[0011] The second aspect of the present invention provides the application of the weakly virulent strain obtained by the above attenuation method in the preparation of vaccines.

[0012] Specifically, the preparation method of the vaccine includes: inoculating the weakly virulent strain into LMH cells for suspension culture, mixing the antigen with high titer with a heat-resistant freeze-drying protectant to prepare a freeze-dried vaccine.

[0013] Preferably, the titer of the virus solution after culture reaches 10 9.0 TCID 50 / mL or more.

[0014] Preferably, the volume ratio of the antigen to the heat-resistant freeze-drying protectant is (4 - 5):1.

[0015] Through the above technical solutions, the present invention achieves the following beneficial effects: In the present invention, the duck short beak and dwarfism syndrome virus strain is treated in an acidic and alkaline environment to obtain a weakly virulent strain with low virulence and strong immunogenicity, which is inoculated into LMH cells for suspension culture, and the titer of the harvested virus solution can reach 10 9.0TCID 50 / mL. Mix a high - titer antigen with a heat - resistant lyophilized protective agent in proportion to prepare a lyophilized vaccine. After inspection, the vaccine has good safety. The immunized animals are in good mental state and no local or systemic adverse reactions caused by the vaccine occur. After immunization and challenge, 100% immune protection effect can be produced in the immunized groups. Detailed implementation manners

[0016] The following details the specific implementation manners of the present invention. It should be understood that the specific implementation manners described herein are only for explaining and illustrating the present invention and are not used to limit the present invention.

[0017] Example 1: Screening and attenuation of duck short beak and dwarf syndrome virus (1) Screening of candidate virulent strains Diseased ducks with typical duck short beak and dwarf syndrome that broke out in a Cherry Valley duck farm in Jining City, Shandong Province were aseptically dissected, and their spleen, kidney and other diseased materials were used for virus isolation with healthy non - immunized Cherry Valley duck embryos; through comparative analysis of its DNA sequence and amino acid sequence with the classical GPV and the published duck short beak and dwarf syndrome sequences. The results showed that a typical duck short beak and dwarf syndrome virus was successfully isolated and named SDJH strain.

[0018] (2) Attenuation of the virus strain The harvested virus strains were attenuated under acidic conditions and alkaline conditions respectively. First, they were placed at pH 4.5 - 5.5 for 4 hours under acidic conditions, then the pH was adjusted to alkaline conditions of 9.0 - 10.0 and placed for 4 hours, and then single - colony cloning was carried out. Strains with low virulence and good immunogenicity were screened through virulence and immunogenicity determination; then the strain was subjected to acidic - condition domestication - alkaline - condition domestication - single - colony - safety screening - potency screening, and then acidic - condition / alkaline - condition domestication. After 5 rounds of cyclic domestication and screening in this way, a duck short beak and dwarf syndrome virus attenuated strain with good safety and high immune potency was obtained, named SDJH01 strain, as the production strain of the live vaccine against duck short beak and dwarf syndrome of the present invention.

[0019] Example 2: Preparation and application of the live vaccine against duck short beak and dwarf syndrome (1) Virus propagation Using the suspension culture technology of a jet - type bioreactor, the attenuated strain of duck short - beak and dwarf syndrome virus obtained in Example 1 was cultured with disposable discardable paper - sheet carriers for LMH cell suspension culture as the matrix. To obtain a high - titer antigen, the reactor rotation speed, initial inoculation density, cell culture days, virus inoculation amount, serum content in the maintenance fluid after virus inoculation, and virus liquid harvest time were explored. The results showed that when the bioreactor rotation speed was 40 rpm and the dissolved oxygen value was not less than 50%, the oxygen - containing conditions required for cell growth could be ensured; the total initial cell inoculation on the 200g paper - sheet carrier was (1 ± 0.2)×10 9 cells, with DMEM / F - 12 culture medium containing 10% fetal bovine serum, and the pH maintained at 7.0 - 7.2, the cells could grow into a good monolayer after 6 days of culture at 37°C; the virus was inoculated at an MOI of 0.1, the serum content in the maintenance fluid was 2%, and all the virus liquid was harvested 94 - 98 hours after virus inoculation. The titer of the harvested virus liquid could reach 10 9.0 TCID 50 / mL. While the highest titer obtained by flask culture was only 10 8.4 TCID 50 / mL. This shows that higher virus production can be obtained by the proliferation of duck short - beak and dwarf syndrome virus on LMH cells in suspension culture, and this suspension process is feasible.

[0020] (2)Vaccine formulation Take 2 mL of the harvested antigen and mix it with 0.5 mL of heat - resistant freeze - drying protectant (the heat - resistant freeze - drying protectant in Example of CN114984232B) in proportion, and prepare the vaccine (1000 birds / bottle) by freeze - drying according to the determined freeze - drying curve.

[0021] (3)Inspection Safety: Dilute the vaccine to 10 birds / mL, immunize 10 7 - day - old ducklings, and subcutaneously inject 1.0 mL into the neck of each. Keep them in isolation and observe daily for 14 days. The mental state should be good, and no local or systemic adverse reactions caused by the vaccine should occur.

[0022] Potency test: Dilute the vaccine to 1 bird / mL and choose one of the following methods.

[0023] ① Serological method: Immunize 10 7 - day - old ducklings, subcutaneously or intramuscularly inject 1 mL of the vaccine into each, and set up 5 ducks inoculated with an equal amount of PBS as the control group. Keep them in isolation under the same conditions. 21 days after immunization, collect blood respectively, separate the serum, and measure the neutralizing antibody titer. The neutralizing antibody titer of the control group should not be higher than 1:2, and the geometric mean of the serum neutralizing antibody titer of the immunized group should not be lower than 1:16.

[0024] ②Immunization and challenge method: Ten 7-day-old ducklings were immunized by subcutaneous or intramuscular injection of 1 mL of the vaccine each. Another five were inoculated with an equal amount of PBS as the control group and were raised in isolation under the same conditions. 21 days after immunization, all ducks were injected with 0.2 mL of duck short beak and dwarfism syndrome virus (containing the SDJH strain with 1000 ELD 50 . After challenge, they were continuously observed for 14 days. All ducks in the control group should show symptoms (see Note 1), and all ducks in the immunized group should be protected (see Note 2).

[0025] Note 1: Criteria for judging the onset of duck short beak and dwarfism syndrome in Cherry Valley ducks 1. Death or paralysis; 2. The large tongue or the beak length has a negative deviation of 15% or more from the average beak length of the healthy control group; 3. Dwarfism or the body weight has a negative deviation of 20% or more from the average body weight of the healthy control group; If any one of the above three items is met, it is judged as having onset.

[0026] Note 2: Criteria for judging the protection of duck short beak and dwarfism syndrome in Cherry Valley ducks 1. Healthy and alive; 2. The negative deviation of the beak length from the average beak length of the healthy control group is within 15%; 3. The negative deviation of the body weight from the average body weight of the healthy control group is within 20%; All three of the above items need to be met simultaneously to be judged as protected.

[0027] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0028] In addition, it should be noted that, among the various specific technical features described in the above specific embodiments, they can be combined in any appropriate manner without contradiction. To avoid unnecessary repetition, the present invention will not separately describe various possible combination methods.

[0029] Furthermore, any combination can be made between various different embodiments of the present invention as long as it does not violate the idea of the present invention, and it should also be regarded as the content disclosed by the present invention.

Claims

1. A method for attenuating duck short beak and dwarfism syndrome virus, characterized in that, It includes the following steps: (1) Domesticate the virulent strain of duck short beak and dwarfism syndrome virus successively under acidic conditions and alkaline conditions; (2) Conduct single colony cloning on the strain treated in step (1), and screen out the strain with good safety and immunogenicity through virulence and immunogenicity determination; (3) Repeat steps (1) and (2) several times to obtain a weakly virulent strain of duck short beak and dwarfism syndrome virus with good safety and high immune efficacy.

2. The attenuation method according to claim 1, wherein In step (1), the screening method for the virulent strain of duck short beak and dwarfism syndrome virus is as follows: Diseased ducks with typical duck short beak and dwarfism syndrome that broke out in a Cherry Valley duck farm in Jining City, Shandong Province are aseptically dissected, and spleen, kidney and other diseased materials are taken for virus isolation using healthy non-immunized Cherry Valley duck embryos; By comparing and analyzing its DNA sequence and amino acid sequence with the classical GPV and the published duck short beak and dwarfism syndrome sequence, a typical duck short beak and dwarfism syndrome virus is isolated.

3. The attenuation method according to claim 1, wherein In step (1), the acidic condition domestication is to place it at pH 4.5 - 5.5 for 4 - 5 hours; and / or The alkaline condition domestication is to place it at pH 9.0 - 10.0 for 4 - 5 hours.

4. The attenuation method according to claim 1, wherein In step (3), repeat steps (1) and (2) 4 - 6 times. Application of the weakly virulent strain obtained by the attenuation method described in any one of claims 1 to 4 in the preparation of a vaccine.

6. The application according to claim 5, wherein The preparation method of the vaccine includes: inoculating the weakly virulent strain into LMH cells for suspension culture, mixing the antigen with high titer with a heat-resistant freeze-drying protectant to prepare a freeze-dried vaccine.

7. The application according to claim 6, characterized in that, The virus titer in the virus solution reaches 10 9.0 TCID 50 / mL or higher.

8. The application according to claim 6, wherein, The volume ratio of the antigen to the heat-resistant freeze-drying protectant is (4 - 5):1.