Chicken source tembusu virus and egg drop syndrome virus bivalent vaccine and application thereof

Abstract

The invention provides a chicken source tembusu virus and egg drop syndrome virus bivalent vaccine, the chicken source tembusu virus is a chicken source tembusu virus SD01 strain, and the preservation number is CCTCC (China Center For Type Culture Collection) NO: V202472. The invention also provides a preparation method of the chicken source tembusu virus strain. The bivalent vaccine can achieve symptomatic prevention on chicken flocks infected with tembusu virus diseases, can effectively control occurrence of egg drop syndromes, and is good in safety, good in immune effect and high in protection rate.

Description

Technical Field

[0001] The present invention belongs to the field of veterinary vaccines, and specifically relates to a chicken-derived Tembusu virus and egg production decline syndrome virus combined vaccine. Background Art

[0002] Tembusu virus (TMUV) belongs to the Flaviviridae family and is a new type of flavivirus in the Entay virus group. Since 2010, it has successively broken out in duck-raising areas such as Zhejiang, Fujian, Anhui, and Shandong in my country, mainly infecting laying ducks, breeder ducks, and some geese. After infection, the main symptoms are loss of appetite, a sharp drop in egg production, and high fever. The incidence rate is as high as 90%, which seriously affects the development of duck and goose farming. With the spread of the virus, since 2023, chickens in many chicken farms in Shandong and Guangdong have also shown clinical symptoms such as fever, loose stools, and decreased egg production. The autopsy of the affected chickens showed pathological changes such as follicular degeneration, hemorrhage, and spleen enlargement. It was identified as Tembusu virus infection. It can be seen that chicken-derived Tembusu virus also has great potential harm to the poultry industry. Egg Drop Syndrome (EDS) is a viral infectious disease that mainly affects laying hens. It is caused by Egg Drop Syndrome Virus (EDSV), which belongs to the genus Adenovirus of avian origin and is a double-stranded DNA virus. Egg drop syndrome is characterized by a sudden drop in egg production and a significant increase in the number of soft-shelled eggs, thin-shelled eggs and deformed eggs. It is a disease that needs to be prevented and controlled in the poultry industry.

[0003] In order to prevent these two diseases that cause a decrease in egg production in laying hens, we urgently need to develop a bivalent vaccine against these two viruses. However, the currently available Tembusu vaccines are mainly targeted at the duck-derived Tembusu virus, and no chicken-derived Tembusu virus strains and vaccines have been reported. Summary of the invention

[0004] In view of this, the present invention aims to overcome the defects in the prior art and provide a chicken-derived Tembusu virus and egg drop syndrome virus bivalent vaccine.

[0005] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0006] A chicken-origin Tembusu virus and egg drop syndrome virus bivalent vaccine, wherein the chicken-origin Tembusu virus is the chicken-origin Tembusu virus SD01 strain, and the preservation number is: CCTCC NO: V202472.

[0007] Preferably, the preparation method of the chicken-derived Tembusu virus strain is:

[0008] 1) The isolated chicken-derived Tembusu virus was blindly propagated on chicken embryos for three generations;

[0009] 2) Plaque purification on primary chick embryo fibroblasts;

[0010] 3) Adhesive culture on fibroblasts and chicken hepatoma cells, with five alternating passages;

[0011] 4) Continue to subculture on chicken liver cancer cell suspension cells to obtain a stable subculture strain.

[0012] Preferably, the vaccine is chicken-derived Tembusu virus 10 7.25 TCID 50 / 0.1mL.

[0013] Preferably, the egg production decline syndrome virus titer for the vaccine is 10 6.75 TCID 50 / 0.1mL.

[0014] Preferably, the vaccine inactivator is BEI inactivator.

[0015] Preferably, the inoculated cell density of the chicken liver cancer cell suspension culture in step 4) is 2.0×10 6 ~3.0×10 6 Pieces / mL.

[0016] Preferably, the volume ratio of chicken-derived Tembusu virus, egg drop syndrome virus and vaccine adjuvant in the bivalent vaccine is 2:3:1.

[0018] Application of the combined vaccine of chicken-derived Tembusu virus and egg drop syndrome virus in the preparation of egg drop syndrome vaccines and drugs.

[0019] Compared with the prior art, the present invention has the following advantages:

[0020] The invention prepares a bivalent inactivated vaccine by adding EDSV virus liquid to a newly isolated chicken-source Tembusu virus liquid. The prepared vaccine can achieve symptomatic prevention of Tembusu virus disease infection in chickens, and can effectively control the occurrence of egg drop syndrome, with good safety, good immune effect and high protection rate.

[0021] This product uses suspension cells to propagate chicken Tembusu virus. Compared with the traditional propagation method in chicken and duck embryos, the operation procedure is simple, the cost is low, and the exogenous virus infection is effectively controlled, reducing production risks. Compared with propagation on LMH adherent cells, LMH suspension cell propagation does not require serum, and the use of bioreactors for suspension cell propagation can propagate a large amount of venom at one time, which can greatly save a lot of time and reagent costs.

[0022] The present invention utilizes a certain proportion of BEI to inactivate the virus liquid. Compared with the traditional formaldehyde inactivation, the inactivation effect is better and the stress on the chickens is less.

[0023] The chicken-origin Tembusu virus SD01 strain was preserved on September 28, 2024. The preservation address is: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. The preservation unit is: China Center for Type Culture Collection, and the preservation number is CCTCC NO: V202472. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the genetic evolutionary tree analysis diagram of the strain. DETAILED DESCRIPTION

[0025] The following specific descriptions are exemplary and are intended to provide further explanation of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements fall within the protection scope of the present invention.

[0026] The test methods used in the following experimental examples are conventional methods unless otherwise specified.

[0027] Example 1: Preparation of chicken-derived Tembusu virus solution and EDSV

[0028] (1) Using the E protein gene sequence of Tembusu virus (TMUV-JS06 strain) in GenBank (GenBank sequence number: KR869106), Primer5.0 software was used to design upstream and downstream primers (F: 5 upstream and downstream primers designed (the addition of a fixed agent can significantly improve the biphasic adjuvant; R: 5 upstream and downstream primers designed (the addition of a fixed agent can show CA-3'). The diseased chicken ovary material was added with physiological saline at a ratio of 1:3 and then ground to obtain the diseased material virus liquid. PCR identification was performed using a commercially available RNA extraction kit and PCR kit to identify and isolate the related strains from the chicken ovaries suspected of being infected with Tembusu virus. The genetic evolution tree of the strains was analyzed, and the specific results are shown in the table. Figure 1 .

[0029] (2) After blind passage on chicken embryos for three generations, the chicken-derived Tembusu virus could be stably propagated on chicken embryo primary fibroblasts (CEFs) after plaque purification and five rounds of alternating passage on CEFs and chicken hepatoma cells (LMHs, adherent).

[0030] (3) Exploration of the culture conditions of chicken Tembusu virus LMH suspension cells: 1) Determine the inoculation ratio to be MOI = 2 and change the cell density to 1.0 × 10 6 ~4.0×10 6The cell density of chicken Tembusu virus LMH suspension cell culture was 2.0×10 6 ~3.0×10 6 10 cells / mL; 2) Determine the cell density to be 2.0×10 6 The inoculation ratio was adjusted according to the MOI value of 1 to 5, and the appropriate inoculation ratio was determined to be MOI = 2 or 3; then the chicken-derived Tembusu virus strain that could be stably propagated on LMH suspension cells was obtained, and the venom was propagated for subsequent experiments. The virus titer was 10 7.25 TCID 50 / 0.1mL. The strain used for the preparation of chicken-derived Tembusu virus vaccine was named as chicken-derived Tembusu virus SD01 strain and was preserved on September 28, 2024 at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province. The preservation unit is China Center for Type Culture Collection, and the preservation number is CCTCC NO: V202472.

[0031] (4) EDSV was inoculated into well-growing LMH monolayer adherent cells for virus propagation. The inoculation doses were 0.5%, 1%, 2%, and 4%. Finally, it was found that EDSV could proliferate well when the inoculation dose on LMH monolayer adherent cells was 0.5% to 1%. The virus liquid that can be used for vaccine preparation was obtained, and the virus titer was 10 6.75 TCID 50 / 0.1mL.

[0032] Example 2: Preparation of a dual inactivated vaccine

[0033] (1) Antigen inactivation: 0.5% BEA was mixed with 0.5% NaOH, and the mother solution of 0.5% BEI was obtained by cyclization for subsequent inactivation. Four concentrations of BEI solutions, 0.01%, 0.04%, 0.08%, and 0.16%, were added to chicken Tembusu virus venom and EDSV for inactivation. The inactivation test was performed after 24 hours of inactivation. The results showed that BEI concentrations of 0.01% to 0.16% could effectively inactivate. Considering the cost and subsequent side effects, the optimal inactivation dose was set at 0.01%. The specific results are shown in Table 1.

[0034] Table 1 BEI inactivation results

[0035]

[0036]

[0037] Note: “—” indicates completely inactivated, “ indicates not inactivated.

[0038] (2) Emulsification: Mix 94 parts by volume of white oil for injection and 6 parts by volume of Siben-80, add 2 parts by mass of aluminum stearate, and stir until transparent. Sterilize under high pressure and cool for later use. This is the oil phase.

[0039] The chicken-derived Tembusu virus solution and EDSV virus solution inactivated in step (1) are mixed at a ratio of 1:1 / 2:3 / 1:2 / 1:3, respectively, and placed in a sterile container. Sterilized Tween-80 is added at a ratio of 4% (V / V) of the virus solution volume, and the mixture is shaken thoroughly until the Tween-80 is completely dissolved, thereby obtaining the aqueous phase.

[0040] Take 2 volumes of oil phase and place them in the oil phase tank, start the motor to stir slowly, then slowly add 1 volume of water phase, after adding, transfer to the emulsification tank, and emulsify at 2800 rpm for 40 minutes to obtain. The specific ratio is shown in Table 2. The relevant vaccine was obtained for subsequent experiments.

[0041] Table 2 Ratio of two viruses in vaccine

[0042] Group Amount of chicken-derived Tembusu virus added EDSV addition amount Mixing ratio with white oil Vaccine 1 1 serving 1 serving 1:1 Vaccine 2 2 servings 3 servings 1:1 Vaccine 3 1 serving 2 servings 1:1 Vaccine 4 1 serving 3 servings 1:1

[0043] Example 3 Immunity Test

[0044] Fifty laying hens aged 90 days were randomly divided into five groups. After one day of adaptation, the following operations were performed:

[0045] Group 1: Set as vaccine group 1, 1.0 mL of vaccine 1 was injected intramuscularly to each feather;

[0046] Group 2: set as vaccine group 2, 1.0 mL of vaccine 2 was injected intramuscularly into each feather;

[0047] Group 3: set as vaccine group 3, 1.0 mL of vaccine 3 was injected intramuscularly into each feather;

[0048] Group 4: set as vaccine group 4, 1.0 mL of vaccine 4 was injected intramuscularly into each feather;

[0049] Group 5: Set as blank control group, each feather was injected intramuscularly with 1.0 mL of normal saline;

[0050] A booster immunization was performed 21 days after the above immunization. Blood samples were collected weekly after the second immunization to detect the antibody level in the serum (see Table 3). The antibody levels of both viruses were evaluated by the neutralization test method. In groups 1 to 4, the serum titer gradually increased over time and eventually remained in a stable range. Among them, group 2 had the best ratio effect, and group 3 was preferred for subsequent tests.

[0051] Table 3 Comparison of serum antibody titers of two viruses with different ratios

[0052]

[0053] Example 4 Comparative test with formaldehyde inactivated vaccine

[0054] Thirty laying hens aged 120 days were randomly divided into three groups. After one day of adaptation, the following operations were performed:

[0055] Group 1: Set as vaccine 2 group, 1.0 mL of vaccine 2 was injected intramuscularly into each feather;

[0056] Group 2: set as the formaldehyde inactivated vaccine group, each feather was intramuscularly injected with 1.0 mL of formaldehyde inactivated vaccine;

[0057] Group 3: Set as blank control group, each feather was injected intramuscularly with 1.0 mL of normal saline;

[0058] As can be seen from Table 4, the BEI inactivated vaccine group 2 had local redness and swelling reactions only on the first day after immunization, which affected the egg production of laying hens; while the formaldehyde inactivated group still had local reactions on the third day after immunization, and still had an impact on the egg production of laying hens on the fifth day.

[0059] Table 4 Safety test results of formaldehyde-inactivated seedlings

[0060]

[0061] Note: "—" indicates no abnormality, "+" indicates redness or swelling at the injection site or significant impact on egg production, etc.

[0062] Example 5: Challenge test

[0063] 80 laying hens aged 90 days were randomly divided into 6 groups. After one day of adaptation, the following operations were performed:

[0064] Group 1: Set as the chicken-derived Tembusu virus challenge control group, each bird was injected intramuscularly with 1.0 mL of normal saline, a total of 10 birds;

[0065] Group 2: 10 birds in total were given an intramuscular injection of 1.0 mL of normal saline per bird as the EDSV challenge control group.

[0066] Group 3: set as the commercially available Tembusu virus vaccine group, each bird was intramuscularly injected with the prescribed amount of commercially available Tembusu virus inactivated vaccine, a total of 10 birds;

[0067] Group 4: set as the traditional formaldehyde inactivated vaccine group, each bird was intramuscularly injected with 1.0 mL of the traditional formaldehyde inactivated bivalent vaccine, a total of 20 birds; Group 5: set as the vaccine 2 group, each bird was intramuscularly injected with 1.0 mL of the BEI inactivated bivalent vaccine, a total of 20 birds;

[0068] Group 6: Set as the healthy control group without any treatment.

[0069] After 21 and 42 days of immunization, chickens in groups 1 / 3 / 4 / 5 were injected intramuscularly with chicken-derived Tembusu virus (10 3.00 ID 50) were used for challenge, and chickens in groups 2 / 4 / 5 were injected intramuscularly with EDSV virus (10 3.00 ID 50 ) were used to challenge the virus, and the incidence and protection rate of each group were calculated. The results showed that when the virus was challenged 21 days after immunization, the incidence of chickens in the control group challenged with chicken-derived Tembusu virus was 90%, and the protection rate of EDSV vaccine group 2 could reach 95%, the highest among all groups, and two diseases could be prevented at the same time; when the virus was challenged 42 days after immunization, the incidence of chickens in the control group challenged with chicken-derived Tembusu virus was 80% to 90%, and the protection rate of the mixture group could reach 100%, the highest among all groups (see Table 5 and Table 6).

[0070] Table 5 Comparison of morbidity and protection rate 21 days after immunization

[0071]

[0072]

[0073] Note: “—” indicates no abnormality, “None” indicates that this item has not been conducted or there is no data for this item;

[0074] Table 6 Comparison of morbidity and protection rate 42 days after immunization

[0075]

[0076] Note: “—” indicates no abnormality, “None” indicates that this item has not been conducted or there is no data for this item;

[0077] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A chicken-derived Tembusu virus and egg drop syndrome virus combined vaccine, characterized in that: The chicken-origin Tembusu virus is the chicken-origin Tembusu virus SD01 strain, and its preservation number is: CCTCC NO: V202472.

2. The chicken-derived Tembusu virus and egg drop syndrome virus bivalent vaccine according to claim 1, characterized in that: The preparation method of the chicken-derived Tembusu virus strain is as follows: 1) The isolated chicken-derived Tembusu virus was blindly propagated on chicken embryos for three generations; 2) Plaque purification on primary chick embryo fibroblasts; 3) Adhesive culture on fibroblasts and chicken hepatoma cells, with five alternating passages; 4) Continue to subculture on chicken liver cancer cell suspension cells to obtain a stable subculture strain.

3. The chicken-derived Tembusu virus and egg drop syndrome virus bivalent vaccine according to claim 1, characterized in that: The vaccine is made of chicken-derived Tembusu virus 10 7.25 TCID 50 / 0.1mL.

4. The chicken-derived Tembusu virus and egg drop syndrome virus bivalent vaccine according to claim 1, characterized in that: The vaccine uses an egg production decline syndrome virus with a titer of 10 6.75 TCID 50 / 0.1mL.

5. The chicken-derived Tembusu virus and egg drop syndrome virus combined vaccine according to claim 1, characterized in that: The vaccine inactivator is BEI inactivator.

6. The chicken-derived Tembusu virus and egg drop syndrome virus bivalent vaccine according to claim 2, characterized in that: The inoculated cell density of the chicken liver cancer cell suspension culture in step 4) is 2.0×10 6 ~3.0×10 6 Pieces / mL.

7. The chicken-derived Tembusu virus and egg drop syndrome virus bivalent vaccine according to claim 1, characterized in that: The volume ratio of the chicken-origin Tembusu virus, the egg drop syndrome virus and the vaccine adjuvant in the bivalent vaccine is 2:3:

1.

8. Use of the chicken-derived Tembusu virus and egg drop syndrome virus combined vaccine as claimed in claim 1 in the preparation of egg drop syndrome vaccines and drugs.

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