Bovine herpesvirus type 4 virus strain and application thereof

Abstract

The invention discloses a bovine herpesvirus type 4 virus strain and application thereof, the bovine herpesvirus type 4 virus strain is a bovine herpesvirus type 4 CB strain, and is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC No.45255, and the preservation date is August 10, 2022. The bovine herpesvirus type 4 inactivated vaccine is safe in application and has a good immune protection effect on bovine herpesvirus type 4 diseases caused by the bovine herpesvirus type 4. The immunized cattle can be protected for at least four months.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to an application of bovine herpesvirus type 4 (BoHV-4), and a vaccine or diagnostic product developed using the virus. Background Art

[0002] Bovine herpesvirus type 4 is a virus that infects cattle and belongs to the family Herpesviridae. The virus can cause a variety of diseases, including respiratory diseases, mastitis, and reproductive tract infections. BoHV-4 infection is widely present in cattle herds in many countries and regions around the world, seriously affecting the health of cattle and the economic benefits of the livestock industry. At present, there is a lack of effective prevention methods, and existing control measures mainly rely on management measures and supportive treatments, which can neither completely eliminate the virus nor effectively prevent the occurrence and spread of the disease.

[0003] Given the above background, the development of a vaccine against BoHV-4 is urgently needed. An effective vaccine can not only prevent the occurrence of the disease and reduce economic losses, but also improve animal welfare and help protect public health from animal-borne diseases. However, there is no relevant vaccine on the market in my country.

[0004] In view of the deficiencies in the prior art, the present invention provides a vaccine based on bovine herpes virus type 4, which can effectively stimulate the body's immune response, thereby preventing diseases caused by BoHV-4. The present invention also provides a method for preparing a vaccine using the virus, and a method for using the vaccine for disease prevention. Summary of the invention

[0005] The purpose of the present invention is to provide a bovine herpesvirus type 4 isolated strain and application thereof.

[0006] The bovine herpesvirus type 4 isolated strain of the present invention is a bovine herpesvirus type 4 CB strain, and its classification name is bovine herpesvirus type 4. It is deposited in the General Microbiology Center of China Microorganism Culture Collection, with a deposit number of CGMCC No.45255, a deposit date of August 10, 2022, and a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, General Microbiology Center of China Microorganism Culture Collection, Postal Code 100101.

[0007] The object of the present invention is to provide a new BoHV-4 virus strain and application thereof in vaccine preparation to achieve effective prevention of diseases caused by BoHV-4.

[0008] (a) Characteristics of the virus strain: The BoHV-4 virus strain involved in the present invention has high safety and good immunogenicity.

[0009] The virus strain was obtained through a specific adaptive culture method, retaining key antigenic properties that can stimulate the immune system response while reducing pathogenicity to the host.

[0010] (b) Vaccine preparation: The BoHV-4 virus strain provided by the present invention is selected and propagated by cell culture technology.

[0011] According to the needs of disease prevention, the virus strain is selected to be inactivated and mixed with a suitable adjuvant to prepare a vaccine.

[0012] The vaccine can be used to prevent diseases caused by BoHV-4 by injection or other appropriate routes of administration.

[0013] Compared with the prior art, the present invention achieves the following beneficial technical effects: Through the application of the virus strain and vaccine provided by the present invention, diseases caused by BoHV-4 can be effectively prevented, the disease incidence rate of cattle can be reduced, and the economic benefits of the breeding industry can be improved.

[0014] The vaccine of the invention has high safety and good immune effect, and provides a new solution for the control of bovine herpes virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The bovine herpesvirus type 4 virus CB strain of the present invention causes MDBK cell lesions; the right side shows MDBK cells that produce cytopathic effects; the left side shows normal MDBK cells; Figure 2 The invention provides PCR detection of bovine herpesvirus type 4 CB strain; gB gene amplification result, wherein the order of gel electrophoresis loading is: lane 1: negative control; lanes 2-6: different generations of bovine herpesvirus type 4; M: 2K plus Marker, from top to bottom are 5000bp, 3000bp, 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp. DETAILED DESCRIPTION

[0016] The following embodiments are merely exemplary and do not constitute any limitation to the scope of the present invention. The details and forms of the technical solutions 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 are within the scope of protection of the present invention.

[0017] The methods used in the present invention can adopt the methods commonly used in the field of virus research, and are not limited to the specific description of the embodiments of the present invention. The experimental methods used in the following embodiments are conventional methods unless otherwise specified. The reagents, materials, etc. used in the following embodiments, unless otherwise specified, can be obtained from commercial channels.

[0018] The reagents and materials used in the following examples are as follows: CO2 incubator purchased from Thermo; high-speed desktop centrifuge purchased from Shanghai Anting Scientific Instrument Factory; gel imaging system Tanon 1600R purchased from Beijing Yuanpinghao Biotechnology Co., Ltd.; PCR instrument purchased from Eppendorf; nucleic acid protein electrophoresis instrument purchased from Beijing Liuyi Instrument Factory; Trans 2K plusMarker, viral DNA / RNA extraction kit, 2×EasyTaq® PCR SuperMix purchased from Beijing Quanshijin Biotechnology Co., Ltd.; 2×MDBK (Madin-Darby Bobine Kidney cell) cells purchased from China Veterinary Drug Administration; DMEM culture medium and 0.25% Trypsin-EDTA (1×) pancreatic enzyme purchased from Gibco; fetal bovine serum purchased from Inner Mongolia Jinyuankang Bioengineering Co., Ltd.

[0019] Example 1 Isolation and Identification of Bovine Herpesvirus Type 4 1.1 Isolation of bovine herpesvirus-4 1.1.1 Treatment of diseased materials The nasal swabs collected from cattle suspected of having herpesvirus type 4 were placed in culture medium (DMEM containing 1000 units / ml penicillin and 1000 μg / ml streptomycin) and stored at 4°C overnight. Then centrifuge at 12000 r / min for 10 min, take the supernatant and filter it with a 0.22μm microporous filter membrane for sterilization, and store it below -70°C for later use.

[0020] 1.1.2 Virus isolation and passaging Inoculate the supernatant after the above treatment into a monolayer of MDBK cells in the logarithmic growth phase, adsorb at 37°C for 2 hours, then pour off the liquid, add DMEM maintenance solution (DMEM containing 200 units / ml of penicillin, 200 μg / ml of streptomycin and 10 ml / L of serum), culture in a 37°C, 5% CO2 incubator, and observe the cytopathic effect (CPE) every day. When 70% to 80% of the cells show CPE, harvest the virus. If the cells do not show CPE, blindly pass it after 5 days. When the cells show lesions, freeze and thaw the harvested culture at -80°C twice, and store it at -80°C for later use. The isolated virus can produce typical cytopathic effect (CPE) in MDBK, see Figure 1 .

[0021] 1.2 PCR Identification of Bovine Herpesvirus-4 Virus 1.2.1 Virus PCR detection The isolates were identified by PCR using primers for amplification of the gB gene of bovine herpesvirus type 4. The primer sequences are shown in Table 1 below. The amplified product was expected to be 615 bp.

[0022] Table 1 BPIV3 PCR primer sequences and amplified fragment sizes

[0023] Sample nucleic acid was extracted using a commercial viral DNA / RNA extraction kit. PCR reaction was performed in a 25 μL system: 2×EasyTaq® PCR SuperMix 12.5 μL, upstream and downstream primers 1 μL each, template DNA 2.0 μL, ddH2O 6.8 μL.

[0024] Reaction conditions: 95℃ 5 min; 94℃ 1mins, 54℃ 1min, 72℃ 3mins, 35 cycles; 72℃ 10min. PCR products were stored at 4℃. Take 5μL of the reaction product, add an appropriate amount of loading buffer, mix well, and electrophoresed in 1% agarose gel at 120V for 30 min to observe the results. The results showed that a specific band of about 615bp was amplified, and the PCR product was sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing analysis. The sequencing results were compared with the sequences published by NCBI, and the results showed that the isolate was bovine herpesvirus type 4. See Figure 2 .

[0025] Example 2 Virus subculture and virus titer determination 2.1 Virus proliferation and culture The bovine herpesvirus type 4 CB strain was inoculated into MDBK monolayer cells at an inoculum of 1:1000 and harvested when the CPE reached more than 80%. The harvested virus solution was tested for sterility, exogenous virus, specificity, etc. and then frozen for seed preservation. This method was used for 15 generations, and the virus harvest volume of each generation was at least 200 ml. The harvested virus was stored at -80℃ for future use.

[0026] 2.2 Determination of toxicity titer Take F1 to F15 generations of virus culture and dilute them 10 times with cell maintenance medium, and determine the virus's cell half infection dose (TCID50) according to the method in the appendix of the Chinese Pharmacopoeia of Veterinary Medicine. The highest toxicity titer can reach 107.5TCID50 / 0.1ml.

[0027] Example 3 Preparation of inactivated bovine herpesvirus type 4 vaccine and duration of immunity 3.1 Virus reproduction The basic seed of bovine herpesvirus type 4 CB strain was diluted 1:10 with DMEM cell maintenance medium, and then the diluted virus solution was inoculated into a well-grown MDBK monolayer cell at an MOI of 0.1, and cultured in a cell culture incubator at 37°C with 5% CO2 for 5 days until more than 80% of the cells showed typical CPE, and the virus content was measured to be 107.5TCID50 / 0.1ml, which can be used as an antigen for preparing vaccines.

[0028] 3.2 Inactivation of bovine herpesvirus type 4 virus Add the above virus solution to 10% formaldehyde solution (37% formaldehyde solution is recorded as 100%), so that the final formaldehyde concentration is 0.2%, stir while adding, mix thoroughly, and inactivate at 37°C for 24 hours. After the inactivation is completed, samples are taken for sterility test and inactivation test, and those that pass the test are used for vaccine preparation.

[0029] 3.3 Vaccine preparation: Mix the inactivated bovine herpes virus type 4 solution that has passed the inspection with 605 adjuvant in a ratio of 1:1 and mix thoroughly at 100-300 r / min.

[0030] 3.4 Safety of inactivated bovine herpesvirus type 4 vaccine The vaccine was injected intramuscularly into the neck of five healthy calves aged 1 to 3 months, with 4.0 ml per calves. The calves were observed for 15 consecutive days and no local or systemic adverse reactions due to the injection of the vaccine were observed.

[0031] 3.5 Efficacy and duration of immunity of inactivated bovine herpesvirus type 4 vaccine As shown in Table 2, 10 healthy calves with negative antibodies aged 2 to 6 months were used, of which 5 calves were injected with 2 ml of inactivated bovine herpesvirus type 4 vaccine into the neck muscle, and the immunization was boosted with the same dose 21 days after the immunization. Blood was collected at 14 days, 2 months, 4 months, 6 months, and 8 months after the second immunization to determine the neutralizing antibody titer. Blood was also collected from 5 control calves, and the serum was separated to detect the serum neutralizing antibody titer.

[0032] Table 2 Efficacy of inactivated vaccine of bovine herpesvirus type 4 CB strain in cattle immunization group

[0033] The results of the immunization experiment are shown in Table 3 below. The bovine herpesvirus type 4 CB strain can induce the production of high levels of antibodies, and the immunity can last up to 6 months.

[0034] Table 3 Results of the study on the efficacy of inactivated vaccine of bovine herpesvirus type 4 CB strain

[0035] The above description describes several preferred embodiments of the invention, but as mentioned above, it should be understood that the invention is not limited to the form disclosed herein, and should not be regarded as excluding other embodiments, but can be used in various other combinations, modifications and environments, and can be modified within the scope of the invention concept described herein through the above teachings or the technology or knowledge of the relevant field. The changes and modifications made by those skilled in the art shall be within the scope of protection of the claims attached to the invention without departing from the spirit and scope of the invention.

Claims

1. A bovine herpesvirus type 4 strain, characterized in that It is a bovine herpesvirus type 4 CB strain, which is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number: CGMCC No. 45255.

2. The bovine herpesvirus type 4 strain according to claim 1, characterized in that: The gB structural protein gene sequence of the bovine herpesvirus type 4 strain is the sequence shown in SEQ ID NO.

1.

3. The bovine herpesvirus type 4 strain according to claim 1, characterized in that: The bovine herpesvirus type 4 strain can proliferate well on MDBK cells, and the virus titer can reach up to 108.5TCID50 / ml.

4. The use of the bovine herpesvirus type 4 strain according to any one of claims 1 to 3, characterized in that: Used to prepare inactivated bovine herpesvirus type 4 vaccine preparations.

5. A bovine herpesvirus type 4 inactivated vaccine preparation, characterized in that: The invention relates to a bovine herpesvirus type 4 virus strain comprising the inactivated bovine herpesvirus type 4 virus strain of claim 1.

6. The method for preparing the inactivated bovine herpesvirus type 4 vaccine preparation according to claim 5, characterized in that: It is made by mixing inactivated bovine herpesvirus type 4 virus liquid and vaccine adjuvant.

7. Use of the inactivated bovine herpesvirus type 4 vaccine preparation according to claim 5 for immunizing cattle.

8. The use according to claim 7, characterized in that: Each dose of the bovine herpesvirus type 4 inactivated vaccine preparation contains at least 107.5TCID50 of bovine herpesvirus type 4 strain.

9. The use according to claim 8, characterized in that: The bovine herpesvirus type 4 inactivated vaccine preparation is injected intramuscularly into cattle, and the immunization dose is 2 ml / head.

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