Gumboro disease virus attenuated vaccine strain and construction method and application thereof

Abstract

The application discloses an infectious bursal disease virus attenuated vaccine strain and a construction method and application thereof, and belongs to the technical field of biotechnology. The application constructs a gene mutant strain through a gene mutation and an infectious cloning operation method, performs a synonymous mutation (TTCAGA) on a TTTCGT motif of a vp2 gene in an IBDV virus genome, and rescues the mutant strain. Compared with a parent strain, the ability of the mutant strain to induce cell apoptosis in an early infection stage is significantly reduced, and the proliferation of the virus is effectively promoted. After the mutant strain is inoculated into SPF chickens, the pathological changes of bursa of Fabricius of the SPF chickens in an early infection stage are obviously weaker than those of the chickens infected by the parent strain, and the replication ability of the mutant strain in an animal body is significantly higher than that of the parent strain. The IBDV genome mutation site provided by the application has important significance for the development of a candidate vaccine strain.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to an attenuated vaccine strain of infectious bursal disease virus, its construction method, and its application. Background Technology

[0002] Infectious bursal disease virus (IBDV) is a virus that primarily affects chicks, damaging immune organs such as the bursa of Fabricius. Belonging to the genus *Avibirnavirus* of the family Birnaviridae, this highly contagious virus can be transmitted through air, feces, or contaminated environments. IBDV infection causes severe economic losses to the poultry industry, mainly due to the disruption of the chickens' immune system, reducing their immunity to other diseases and increasing disease incidence. Infected poultry exhibit clinical symptoms such as depression, loss of appetite, and diarrhea, and in severe cases, can lead to mass mortality.

[0003] Currently, IBDV control measures mainly rely on vaccination. However, due to the emergence of IBDV variants, which differ significantly from classic strains in antigenicity and pathogenicity, they can evade the immune protection of commercially available vaccines. Existing vaccines have limited effectiveness against some variant strains. Furthermore, excessively virulent virus strains can severely damage the poultry immune system, reducing the ability of the immune response and further exacerbating economic losses. Therefore, reducing IBDV virulence not only helps improve vaccine efficacy and alleviate immunosuppression but also effectively reduces poultry mortality and production losses.

[0004] VP2 protein is a major structural protein of IBDV, inducing the production of neutralizing antibodies and serving as a primary host protective antigen for IBDV. Positions 253, 279, 284, and 330 of VP2 are recognized virulence sites for IBDV. Mutations at positions 253, 279, and 284 can allow highly virulent IBDV strains that cannot adapt to CEF cell culture to adapt to CEF cells, representing key sites affecting receptor binding and related to cell tropism. However, the key sites related to IBDV-induced apoptosis remain unclear. Summary of the Invention

[0005] [Technical Issues]

[0006] The technical problem to be solved by the present invention is to provide an attenuated vaccine strain for infectious bursal disease virus.

[0007] [Technical Solution]

[0008] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0009] In a first aspect, the present invention provides a polynucleotide having the nucleotide sequence shown in SEQ ID NO.1.

[0010] In a second aspect, the present invention provides an infectious bursal disease virus VP2 protein, the nucleotide sequence of the gene encoding the infectious bursal disease virus VP2 protein being shown in SEQ ID NO.1.

[0011] In a third aspect, the present invention provides a recombinant infectious bursal disease virus, wherein the recombinant infectious bursal disease virus uses infectious bursal disease virus as a parent strain, and the VP2 protein coding gene of infectious bursal disease virus in the genome of the parent strain is replaced with the nucleotide sequence shown in SEQ ID NO.1.

[0012] In one embodiment, the parent strain includes infectious bursal disease virus Lohmann Lx.

[0013] In one embodiment, the recombinant infectious bursal disease virus has reduced virulence compared to the parent strain.

[0014] In a fourth aspect, the present invention provides an immunogenic composition containing the antigen of the recombinant infectious bursal disease virus or its culture as described in the third aspect.

[0015] In one embodiment, the antigens of the recombinant infectious bursal disease virus or its culture include inactivated whole virus antigen, live attenuated whole virus antigen, subunit antigen, synthetic peptide antigen, and / or live vector antigen.

[0016] In one embodiment, the subunit antigen is the infectious bursal disease virus VP2 protein.

[0017] In a fifth aspect, the present invention provides a vaccine comprising the immunogenic composition described in the fourth aspect.

[0018] In one embodiment, the vaccine further includes a pharmaceutically acceptable carrier.

[0019] In a sixth aspect, the present invention also provides a method for reducing the virulence of infectious bursal disease virus, using infectious bursal disease virus as a parent strain, and replacing the infectious bursal disease virus VP2 protein encoding gene on the genome of the parent strain with nucleotides having a sequence as shown in SEQ ID NO.1.

[0020] In a seventh aspect, the present invention also provides the use of the polynucleotide of the first aspect, the infectious bursal disease virus VP2 protein of the second aspect, the recombinant infectious bursal disease virus of the third aspect, the immunogenic composition of the fourth aspect, the vaccine of the fifth aspect, or the method of the sixth aspect in the preparation of a medicament for the prevention and / or treatment of diseases caused by infectious bursal disease virus.

[0021] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] This invention constructs a mutant strain using gene mutation and infectious cloning methods. In the IBDV viral genome, a synonymous mutation (TTCAGA) is performed on the TTTCGT motif of the vp2 gene to rescue the mutant strain. Compared to the parent strain, its ability to induce apoptosis in the early stages of infection is significantly reduced, and it effectively promotes viral proliferation. When inoculated into SPF chickens, the lesions in the bursa of Fabricius of SPF chickens were significantly weaker than those of chickens infected with the parent strain in the early stages of infection, while the replication capacity of the mutant strain in animals was significantly higher than that of the parent strain. The IBDV genomic mutation sites provided by this invention are of great significance for the development of candidate vaccine strains. Attached Figure Description

[0024] Figure 1 RT-PCR identification of the parental strain (WT: IBDV-Lx-WT) and the mutant strain (Mut: IBDV-Lx-Mut422).

[0025] Figure 2 The sequencing results are for the mutation sites: WT: IBDV-Lx-WT, Mut: IBDV-Lx-Mut422.

[0026] Figure 3 IFA identification of the parental strain (WT: IBDV-Lx-WT) and the mutant strain (Mut: IBDV-Lx-Mut422) was performed. The rescued parental and mutant strains were inoculated into DF-1 cells using standard methods and cultured in a 37°C CO2 incubator for 24 h. Indirect immunofluorescence detection was then performed using anti-IBDV-VP4 McAb.

[0027] Figure 4Western blot analysis of viral protein expression in the parental strain (WT: IBDV-Lx-WT) and the mutant strain (Mut: IBDV-Lx-Mut422) was performed. The rescued parental and mutant strains were inoculated into DF-1 cells using standard methods and cultured in a 37°C CO2 incubator for 24 h. Cells were then collected, lysed, and denatured by boiling in water for 8 min with protein loading buffer. Western blot analysis was performed using anti-IBDV-VP4 McAb.

[0028] Figure 5 Replication kinetics of the parental strain (IBDV-Lx-WT) and the mutant strain (IBDV-Lx-Mut422) were analyzed. The parental strain or the mutant strain was infected with MOI = 0.1. Infected cell supernatants were collected at 12, 24, 36, and 48 h post-infection, and their TCID values ​​were titrated. 50 Titer; repeat 3 times, take the average result and plot the viral replication kinetics curve.

[0029] Figure 6 Western blot analysis was performed on the parental strain (WT: IBDV-Lx-WT) and the mutant strain (Mut: IBDV-Lx-Mut422). The parental strain or the mutant strain was infected with an MOI of 0.01. Viral proteins were collected at 12, 18, 24, 36, and 48 hours, denatured by adding protein loading buffer and boiling in water for 8 minutes, and then analyzed by Western blot.

[0030] Figure 7 Cytopathic effects at different time points after infection of cells with the parental strain (WT: IBDV-Lx-WT) and the mutant strain (Mut: IBDV-Lx-Mut422). The parental strain or the mutant strain was infected with MOI=0.01, and cytopathic effects were observed and photographed at 12, 18, 24, 36, and 48 h post-infection.

[0031] Figure 8 Cell viability was measured at different time points after infection with the parental strain (WT: IBDV-Lx-WT) and the mutant strain (Mut: IBDV-Lx-Mut422). The parental strain or mutant strain was infected with MOI = 0.01, and cells were stained with trypan blue at 12, 18, 24, 36, and 48 h post-infection. Cells were then counted using a cell counter, and the percentage of viable cells was recorded.

[0032] Figure 9Early apoptosis was observed 24 hours after infection with the parental strain (WT: IBDV-Lx-WT) and the mutant strain (Mut: IBDV-Lx-Mut422). Cells infected with either the parental strain or the mutant strain at MOI = 0.01 for 24 hours were treated with the Annexin V-FITC apoptosis kit, and apoptosis was analyzed by flow cytometry.

[0033] Figure 10 for Figure 9 The quantitative results are presented as mean ± SD, with data representing three independent replicate experiments. ****, P < 0.0001.

[0034] Figure 11 For muscle hemorrhage, SPF chickens were inoculated with the parental strain (IBDV-Lx-WT) and the mutant strain (IBDV-Lx-Mut422), respectively. They were euthanized 7 days after infection to observe the muscle hemorrhage.

[0035] Figure 12 Images of bursal lesions in different treatment groups.

[0036] Figure 13 The Bursa of Fabricius Index (BBIX) is calculated as follows: BBIX = (weight ratio of chicken bursa in the experimental group to weight ratio of chicken bursa in the control group). When BBIX < 0.7, it is considered a bursa of Fabricius lesion.

[0037] Figure 14 Pathological sections of the bursa of Fabricius and spleen from different treatment groups.

[0038] Figure 15 The viral load in the bursa of Fabricius was measured in different treatment groups. The viral RNA copy number in the bursa of Fabricius on day 7 post-challenge was detected using RT-qPCR. Detailed Implementation

[0039] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation of the invention in any way.

[0040] In this disclosure, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all widely used terms and routine procedures in their respective fields. To better understand this disclosure, definitions and explanations of relevant terms are provided below.

[0041] In this invention, the term "about" or "approximately" should be understood to include all values ​​within the permissible range of measurement error.

[0042] In this invention, the terms "nucleic acid," "nucleic acid molecule," "nucleic acid sequence," "nucleotide sequence," and "polynucleotide" are used interchangeably and refer to polymeric forms of nucleotides (ribonucleotides or deoxyribonucleotides) of any length. Therefore, this term includes, but is not limited to, single-stranded, double-stranded, or mixed-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers comprising, consisting of, or substantially consisting of purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derived nucleotide bases.

[0043] In this invention, the term "culture" refers to different passages of a virus culture, and those skilled in the art know that only minor variations in the gene sequence may occur between different passages.

[0044] In this invention, the term "pharmaceuticalally acceptable carrier" refers to a carrier or diluent in the vaccine composition of this invention that does not impede the biological activity and properties of the compound, other than the infectious bursal disease virus antigen.

[0045] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0046] Example:

[0047] In the following examples, the parent strain is infectious bursal disease virus Lohmann Lx, abbreviated as IBDV-Lx-WT; the constructed attenuated strain (mutant strain) is denoted as IBDV-Lx-Mut422.

[0048] Example 1: Construction of attenuated strain

[0049] (1) Construction of IBDV infectious clonal plasmids

[0050] The primers for site mutation are as follows:

[0051] F:5'-CACTGACTTCAGAGAATACTTCATGGAGGTGGCCG-3';

[0052] R:5'-ATTCTCTGAAGTCAGTGTACTCCCTTGTTGGCCA-3'.

[0053] The Lx virus genome (NCBI accession number AF194428.1) was amplified using the A fragment of the IBDV Lx strain genome as a template. This genome fragment was ligated between the RiboJ and HDVrz sites in the PCAGGS plasmid to obtain plasmid pCAGGS-A. Using pCAGGS-A as a template, site-directed mutagenesis amplification was performed using the primers described above. The PCR amplification product was digested with Dpn I enzyme, gently mixed, and incubated at 37°C for 4 hours. The mixture was then transformed into DH5α competent cells, and single colonies were picked for PCR amplification and sequencing. The sequencing primers (F1 and R1) are as follows:

[0054] F1:5'-TTCGAGCTGATCCCAAATCCTGAACTAGC-3';

[0055] R1:5'-TCTGACGGCACTCTCGAGTTC-3'.

[0056] Plasmids were extracted from strains whose mutation sites were successfully verified by sequencing, and the mutated plasmid was named pCAGGS-mutA. The vp2 gene sequence in the mutated A fragment is shown in SEQ ID NO.1.

[0057] (2) Virus rescue

[0058] DF-1 cells were seeded in vials (DMEM containing 10% FBS). When the cells reached 70-90% confluence, the culture medium was aspirated, and the cells were washed three times with PBS. 5 mL of serum-free culture medium was then added. Lipofectamine was used to... TM Using the 3000 transfection reagent, pCAGGS-A and pCAGGS-mutA were ligated to pCAGGS-B (the Lx virus genome (accession number AF194429.1) amplified using the B fragment of the IBDV Lx strain genome as a template) and then ligated between the RiboJ and HDVrz sites of the PCAGGS plasmid. DF-1 cells were then co-transfected. After 6–8 hours of transfection, the cells were replaced with cell maintenance medium containing 2% FBS and cultured for 3–5 days. Cell status was observed daily, and the virus was harvested when obvious lesions appeared. Cells were frozen and thawed, centrifuged, and the supernatant was collected. The cells were then blindly passaged 3 times to obtain the mutant strain.

[0059] After the rescued virus was passaged 15 times in DF-1 cells, the viral genome was extracted, and the vp2 gene was cloned using RT-PCR (agarose gel electrophoresis results are shown below). Figure 1 As shown in the figure, the amplified products were sequenced. The results showed that the site in the genome was successfully mutated (as shown in the figure). Figure 2 ).

[0060] The parental virus strain (IBDV-Lx-WT) and the mutant strain (IBDV-Lx-Mut422) were inoculated into DF-1 cells and cultured in a 37℃ CO2 incubator for 24 h. The mutant strain was then rescued using indirect immunofluorescence detection with anti-IBDV-VP4 McAb. Results are as follows: Figure 3 As shown, both the parental strain and the mutant strain showed green specific fluorescence in infected cells, while no fluorescence was visible in the normal cell control group.

[0061] The parental IBDV strain (IBDV-Lx-WT) and the mutant strain (IBDV-Lx-Mut422) were inoculated into DF-1 cells and cultured in a 37℃ CO2 incubator for 24 h. Cells were then collected, lysed, and denatured by boiling in water for 8 min with protein loading buffer. Western blotting was performed using anti-IBDV-VP4 McAb. The results are as follows: Figure 4 As shown, the mutant strain infected group expressed VP4 protein.

[0062] Example 2: Detection of Replication Capacity of Mutant Strains

[0063] DF-1 cells were seeded in 6-well plates. After the cells reached a confluent monolayer, the culture medium was discarded, and the cells were washed three times with PBS. The parental strain (IBDV-Lx-WT) and the mutant strain (IBDV-Lx-Mut422) were infected with the cells at an MOI of 0.1. The cells were incubated at 37°C for 2 hours, the supernatant was discarded, and the cells were washed twice with PBS. 2 mL of cell maintenance medium (DMEM containing 2% FBS and 100 IU / mL penicillin antibiotics) was added, and the cells were incubated in a 37°C CO2 incubator. The supernatant from infected cells was collected at 12, 24, 36, and 48 hours post-infection, and TCID values ​​were measured. 50 The experiment was repeated three times, and the average result was used to plot a viral replication kinetics curve. The results are as follows: Figure 5 As shown, the mutant strain has a significantly stronger replication ability than the parent strain.

[0064] DF-1 cells were infected with the parental strain (IBDV-Lx-WT) and the mutant strain (IBDV-Lx-Mut422) at an MOI of 0.01. Viral proteins were collected at 12, 18, 24, 36, and 48 h post-infection. The proteins were denatured by boiling in protein loading buffer for 8 min, and the expression of VP2 and VP4 proteins was detected by Western blotting. Results are as follows: Figure 6 As shown, the expression of VP2 and VP4 proteins increased with increasing challenge time; at 24 h of viral infection, the expression levels of VP2 and VP4 proteins in the parental strain were higher than those in the mutant strain; while at 36 h and 48 h of viral infection, the expression levels of VP2 and VP4 proteins in the parental strain were lower than those in the mutant strain.

[0065] Example 3: Analysis of the ability of viruses to induce apoptosis

[0066] DF-1 cells were infected with the parental strain (IBDV-Lx-WT) and the mutant strain (IBDV-Lx-Mut422) at an MOI of 0.01. After incubation at 37°C for 2 h, the supernatant was discarded, and the cells were washed twice with PBS. 2 mL of cell maintenance medium (DMEM containing 2% FBS and 100 IU / mL penicillin antibody) was added, and the cells were incubated at 37°C. Cytopathic effects were observed at 12, 18, 24, 36, and 48 h post-infection, and trypan blue staining was used. Cell counts were performed using a cell counter, and the percentage of viable cells was recorded. Results are as follows: Figure 7 and Figure 8 As shown, the percentage of viable cells in DF-1 cells at 24, 36, and 48 hours after infection with the mutant strain was significantly higher than that of the parent strain.

[0067] DF-1 cells were seeded in 6-well cell culture plates. When the cells reached 80% confluence, the supernatant was discarded, and the cells were washed three times with PBS. The cells were then infected with an MOI of 0.01 and incubated at 37°C for 2 hours. The supernatant was discarded, and the cells were washed twice with PBS. 2 mL of cell maintenance medium (DMEM containing 2% FBS and 100 IU / mL penicillin antibody) was added, and the cells were incubated at 37°C. 24 hours after infection, the cell culture medium was collected in centrifuge tubes, washed once with PBS, and the cells were digested with trypsin. The cell pellet was collected by horizontal centrifugation at 1000 rpm for 5 minutes. Annexin V-FITC and propidine iodide staining solution were added, and the mixture was gently mixed. The cells were incubated at room temperature in the dark for 15 minutes, then placed on ice. The cells could be resuspended 2-3 times during incubation. The processed samples were then analyzed by flow cytometry. Results are as follows: Figure 9 and Figure 10 As shown, the mutant strain had significantly fewer apoptotic cells in the early stage than the parent strain.

[0068] Example 4: Animal Experiment Evaluation

[0069] Fifteen healthy, two-week-old SPF chickens with no significant weight difference (housed in a negative pressure isolator) were randomly divided into three groups of five chickens each. Two challenge groups were inoculated via eye drops and nasal drops, respectively, with the parental strain (IBDV-Lx-WT) and the mutant strain (IBDV-Lx-Mut422), at a dose of 10... 4.5 TCID 50 In the blank control group, chickens were inoculated with 0.2 mL of DMEM culture medium via eye drops or nasal drops. Seven days after inoculation, the chickens were euthanized. Pathological changes in the spleen, bursa of Fabricius, leg muscles, and pectoral muscles were observed after euthanasia. The bursa of Fabricius and spleen of chickens from each group were photographed and weighed to calculate the bursa of Fabricius index (BBIX). Tissue sections were prepared, and the viral load in the tissues was detected. Results showed that, compared to chickens infected with the parent strain, chickens infected with the mutant strain showed increased hemorrhage in the leg muscles (…). Figure 11) and bursa of Fabricius atrophy ( Figure 12 and 13 The pathogenicity of the mutant strain was significantly reduced compared to that of the parent strain; histopathological sections of the bursa of Fabricius and spleen also showed that the pathogenicity of the mutant strain infecting chickens was significantly reduced compared to that of the parent strain. Figure 14 The results of tissue viral load analysis showed that the mutant strain had a slightly higher replication capacity in animals than the parental strain. Figure 15 ).

[0070] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A polynucleotide, characterized in that, The polynucleotide has the nucleotide sequence shown in SEQ ID NO.

1.

2. The VP2 protein of infectious bursal disease virus, characterized in that, The nucleotide sequence of the gene encoding the VP2 protein of the infectious bursal disease virus is shown in SEQ ID NO.

1.

3. Recombinant infectious bursal disease virus, characterized in that, The recombinant infectious bursal disease virus uses infectious bursal disease virus as the parent strain, and replaces the infectious bursal disease virus VP2 protein encoding gene in the parent strain genome with the nucleotide sequence shown in SEQ ID NO.

1.

4. An immunogenic composition, characterized in that, It contains the antigen of the recombinant infectious bursal disease virus or its culture as described in claim 3.

5. The immunogenic composition according to claim 4, characterized in that, The antigens of the recombinant infectious bursal disease virus or its culture include inactivated whole virus antigen, live attenuated whole virus antigen, subunit antigen, synthetic peptide antigen, and / or live vector antigen.

6. The immunogenic composition according to claim 5, characterized in that, The subunit antigen is the VP2 protein of infectious bursal disease virus.

7. A vaccine, characterized in that, Contains the immunogenic composition according to any one of claims 4-6.

8. The vaccine according to claim 7, characterized in that, The vaccine also includes a pharmaceutically acceptable carrier.

9. A method for reducing the virulence of infectious bursal disease virus, characterized in that, Using infectious bursal disease virus as the parent strain, the gene encoding the VP2 protein of infectious bursal disease virus in the genome of the parent strain was replaced with nucleotides with the sequence shown in SEQ ID NO.

1.

10. The use of the polynucleotide of claim 1, the infectious bursal disease virus VP2 protein of claim 2, the recombinant infectious bursal disease virus of claim 3, the immunogenic composition of any one of claims 4-6, the vaccine of claim 7 or 8, or the method of claim 9 in the preparation of a medicament for the prevention and / or treatment of diseases caused by infectious bursal disease virus.

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