Duck-origin recombinant infectious bronchitis virus and application thereof

Abstract

This invention discloses a duck-derived recombinant infectious bronchitis virus and its applications, belonging to the field of viral isolation and application. The virus was deposited at the China Center for Type Culture Collection on December 26, 2024, with accession number CCTCC NO: V202503. The duck-derived recombinant infectious bronchitis virus of this invention is capable of infecting CEF and DEF cells, is convenient for in vitro culture, and is suitable for the production of high-quality inactivated vaccines with high antigen titers. The duck-derived recombinant infectious bronchitis virus DIBVSD2401 of this invention has excellent immunogenicity, and the inactivated vaccine prepared using the duck-derived recombinant infectious bronchitis virus DIBVSD2401 of this invention has good safety and excellent protective effect.

Description

Technical Field

[0001] This invention relates to the field of virus isolation and application, specifically to a duck-derived recombinant infectious bronchitis virus and its application. Background Technology

[0002] Avian Infectious Bronchitis (IB) is an acute, highly contagious, viral respiratory disease caused by the Infectious Bronchitis Virus (IBV). It is one of the major infectious diseases that seriously threaten the poultry industry. Since its first isolation in 1937, IBV has evolved into nine genotypes, comprising 38 lineages. Genotype I alone includes 30 different viral lineages (named GI-1 to GI-30), while the other genotypes consist of single lineages (GII-1, GIII-1, GIV-1, GV-1, GVI-1, GVII-1, GVIII-1, and GIX-1). In Asia, especially East Asia, the QX (GI-19) lineage is the most prevalent and poses a significant threat to the poultry industry.

[0003] The IBV genome, approximately 27.6 kb in length, is the largest known RNA virus and exhibits high variability. During replication within host cells, the IBV RNA polymerase lacks self-correction capabilities, making the genome highly susceptible to gene mutations, insertions, deletions, and alterations in neutralizing antigen sites, leading to new genotypes and serotypes. Studies have shown that the S protein exhibits the greatest degree of differential variation among the four structural proteins of IBV, and even small mutations (<5%) in the S1 protein can cause antigenic drift, significantly affecting viral host tropism and vaccine efficacy. With the continuous mutation of IBV strains, the number of IBV serotypes and genotypes is increasing, and different serotypes show little or no cross-protection, posing a significant challenge to the diagnosis and control of IBV.

[0004] Therefore, starting with the pathogen, further research into the molecular pathogenesis and viral mutation mechanisms, and the development of corresponding vaccines are key to controlling this infectious disease. The purification and culture of infectious bronchitis virus (IBV) has always been a challenge in virus and vaccine research. Currently, there are very few stable cell lines suitable for in vitro IBV culture. Therefore, obtaining virus strains adapted for in vitro cell culture is crucial for vaccine development and production. Summary of the Invention

[0005] In view of the above-mentioned prior art, the purpose of this invention is to provide a duck-derived recombinant infectious bronchitis virus and its application. This invention isolates a novel infectious bronchitis virus from diseased ducklings, which is a recombinant strain of GI-19 (QX subtype) IBV and duck coronavirus (DcoV). The whole genome sequence, cell tropism, pathogenicity, and immunogenicity of this novel duck-derived recombinant infectious bronchitis virus were investigated, providing a guarantee for the prevention and control of IB.

[0006] Specifically, the present invention relates to the following technical solutions:

[0007] In a first aspect, the present invention provides a duck-derived recombinant infectious bronchitis virus, named duck-derived recombinant infectious bronchitis virus (DIBVSD2401); the virus was deposited on December 26, 2024 at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China, with accession number CCTCC NO:V202503.

[0008] Through recombination with DCoV NC048214 strain and several QX-like strains, the current DIBVSD2401 strain was ultimately formed. Due to gene recombination, the duck-derived recombinant infectious bronchitis virus DIBVSD2401 strain of this invention has the following characteristics compared to existing chicken infectious bronchitis viruses:

[0009] (1) Ducklings showed intestinal infection and growth retardation, and some developed dwarfism; laying ducks showed pathological changes such as decreased egg production, increased deformed eggs and ovarian atrophy.

[0010] (2) It can successfully proliferate in chicken embryo fibroblasts (CEF) and duck embryo fibroblasts (DEF) and cause significant cytopathic effects (CPE).

[0011] In a second aspect, the present invention provides the use of the above-mentioned duck-derived recombinant infectious bronchitis virus in the preparation of a vaccine for the prevention or treatment of avian infectious bronchitis.

[0012] In the above application, the avian infectious bronchitis is caused by duck-derived recombinant infectious bronchitis virus with accession number CCTCC NO:V202503.

[0013] Preferably, the vaccine is an inactivated vaccine or a live attenuated vaccine; more preferably, the vaccine is an inactivated vaccine.

[0014] In a third aspect, the present invention provides a vaccine for the prevention or treatment of avian infectious bronchitis, the vaccine containing an immunologically effective amount of duck-derived recombinant infectious bronchitis virus fluid; the duck-derived recombinant infectious bronchitis virus has the accession number CCTCC NO:V202503.

[0015] Preferably, the viral fluid of duck-derived recombinant infectious bronchitis virus is prepared by the following method:

[0016] The duck-derived recombinant infectious bronchitis virus with accession number CCTCC NO:V202503 was inoculated into duck embryo fibroblasts (DEF) or chicken embryo fibroblasts (CEF). After the duck-derived recombinant infectious bronchitis virus was proliferated and cultured, the cells were broken by freeze-thaw, the supernatant was collected and purified to obtain the viral fluid of duck-derived recombinant infectious bronchitis virus.

[0017] Furthermore, the vaccine also contains adjuvants. Adjuvants are substances that can non-specifically alter or enhance the body's specific immune response to antigens, playing an auxiliary role. Adjuvants can induce a long-term, highly effective specific immune response, improving the body's protective ability, while simultaneously reducing the amount of immune substances required and lowering vaccine production costs. Adjuvants that can be used in this invention include, but are not limited to: aluminum adjuvants, oil emulsion adjuvants, propolis adjuvants, liposome adjuvants, and small peptide adjuvants.

[0018] The beneficial effects of this invention are:

[0019] (1) The duck-derived recombinant infectious bronchitis virus DIBVSD2401 of the present invention has the ability to infect CEF and DEF cells, which is convenient for in vitro culture. Moreover, the duck-derived recombinant infectious bronchitis virus DIBVSD2401 shows lesions on CEF cells early, has an early harvest time, and has a high virus content, making it very suitable for the production of high-quality inactivated vaccines with high antigen titers.

[0020] (2) The duck-derived recombinant infectious bronchitis virus DIBVSD2401 of the present invention has excellent immunogenicity. The inactivated vaccine prepared using the duck-derived recombinant infectious bronchitis virus DIBVSD2401 of the present invention has good safety, excellent protective effect, and long duration of immune protection. Attached Figure Description

[0021] Figure 1 Clinical symptoms of IBV infection in laying ducks include: whitening of eggshells; reduced egg quality; and an increase in the number of deformed, sandy-shelled, and soft-shelled eggs.

[0022] Figure 2The images show the pathological changes in duck embryos after inoculation with the DIBVSD2401 isolate. The leftmost embryo is the control group, which develops normally; the others are the infected embryos, which show obvious dwarfism and curling symptoms.

[0023] Figure 3 This is a schematic diagram of the genome structure of the DIBVSD2401 strain, showing the glycosylation sites on the spike protein (S protein) and its molecular characteristics;

[0024] Figure 4 The results of sequence homology analysis between the DIBVSD2401 strain and 31 major avian coronaviruses in the complete genome and S protein;

[0025] Figure 5 The results of phylogenetic analysis of DIBVSD2401 strain and 31 major avian coronaviruses on the complete genome and S protein;

[0026] Figure 6 Sequence homology analysis of the complete genome, non-structural genes, structural genes, and accessory genes of DIBVSD2401 strain with 37 chicken-derived CIBV strains and 2 DCoV strains;

[0027] Figure 7 Phylogenetic analysis of the S1 gene of DIBVSD2401 strain with 37 chicken-derived CIBV strains and 2 DCoV strains;

[0028] Figure 8 Five possible recombination events in the DIBVSD2401 strain were estimated using the Kimura (two-parameter) distance model.

[0029] Figure 9 The recombinant genome map of DIBVSD2401 generated using the BLAST Ring Image Generator shows the distribution characteristics of the genome sequence;

[0030] Figure 10 The dynamic changes in the RNA copy number of DIBVSD2401 virus in CEF and DEF cells.

[0031] Figure 11 Ducklings infected with DIBVSD2401 showed significant growth retardation at 5 days post-infection (dpi), while ducklings in the control group showed normal growth.

[0032] Figure 12 The figure shows the weight changes of ducklings infected with DIBVSD2401; the horizontal axis unit is d.

[0033] Figure 13 The expression of the virus in different tissues of ducklings infected with DIBVSD2401.

[0034] Figure 14 The clinical manifestations of laying ducks infected with the DIBVSD2401 strain include an increase in thin-shelled eggs, sandy-shelled eggs, broken eggs, and deformed eggs.

[0035] Figure 15 The symptoms of ovarian lesions in laying ducks infected with the DIBVSD2401 strain include follicular congestion, necrosis of newborn follicles, follicular hemorrhage, and follicular liquefaction.

[0036] Figure 16 Analysis of viral load of DIBVSD2401 strain in different tissues of laying ducks.

[0037] Figure 17 After infecting laying ducks with the DIBVSD2401 strain, the egg production rate of the ducks decreased significantly. Detailed Implementation

[0038] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0039] The term "immunely effective dose" refers to the amount of nonvirulent duck recombinant infectious bronchitis virus that elicits an immune response against duck recombinant infectious bronchitis virus.

[0040] To enable those skilled in the art to better understand the technical solutions of this application, the technical solutions of this application will be described in detail below with reference to specific embodiments. Unless otherwise specified, experimental conditions not detailed in the embodiments are generally based on conventional conditions or conditions recommended by the reagent company; reagents, consumables, etc., used in the following embodiments can be obtained commercially unless otherwise specified.

[0041] Example 1: Isolation and identification of duck-derived recombinant infectious bronchitis virus

[0042] 1. Epidemiological investigation:

[0043] Recently, Cherry Valley ducks on several farms in Shandong Province have exhibited symptoms such as diarrhea, stunted growth, and poor weight gain. Particularly in one duck farm, clinical symptoms such as reduced egg production, increased deformed eggs, and whitened eggshells have been observed. Figure 1 This resulted in severe economic losses.

[0044] 2. Pathological sample collection and processing:

[0045] Cloacal swabs were collected from symptomatic ducks. The samples were placed in 1 mL of sterile phosphate-buffered saline (PBS) containing 10% glycerol and shaken. The samples were centrifuged at 12,000 × g for 1 minute at 4°C, and the supernatant was collected. The supernatant was filtered through a 0.45 μm Millipore membrane, and the resulting suspension was used for RNA extraction and virus isolation.

[0046] Total RNA was extracted using TRIzol reagent (Takara, Dalian, China). Reverse transcription was performed according to the Invitrogen kit instructions to synthesize cDNA. PCR amplification was performed using specific primers targeting AIV-H5, H7, H9, novel duck reovirus (NDRV), avian infectious laryngotracheitis virus (ILTV), avian adenovirus (FAdV), Tembusu virus (TMUV), infectious bronchitis virus (IBV), and Newcastle disease virus (NDV).

[0047] The results showed that only infectious bronchitis virus (IBV) was found, while other pathogens such as avian influenza virus (H5, H7, H9), reovirus (NDRV), infectious laryngotracheitis virus (ILTV), adenovirus (FADV), Tembusu virus (TMUV), and Newcastle disease virus (NDV) were not detected in the same specimen.

[0048] 3. Virus isolation and passage:

[0049] To further confirm the virus species, IBV-positive samples were inoculated into the allantoic cavity of 9-day-old SPF chicken embryos. Three passages were performed to confirm virus stability. The allantoic fluid from the third passage was collected and stored at -80°C for later use. The lesion characteristics of the chicken embryos were recorded.

[0050] After the third generation, it was observed that the embryos began to die between the 3rd and 5th days, exhibiting characteristic dwarf embryos (see...). Figure 2 PCR results showed that the third-generation allantoic fluid of the IBV isolate had no hemagglutination activity, ruling out interference from exogenous avian hemagglutinating viruses (such as NDV, AIV, and FADV).

[0051] 4. Virus titer determination:

[0052] The allantoic fluid was serially diluted 10-fold in sterile 0.9% NaCl (10... -2 Up to 10 -7 Each dilution (0.2 mL / embryo) was inoculated into the allantoic cavity of 10-day-old SPF chicken embryos, with 10 embryos used for each dilution. Ten embryos were set up as a negative control and inoculated with 0.2 mL of sterile 0.9% NaCl. They were incubated at 37°C for 5 days, and mortality was recorded every 24 hours until 120 hours after infection.

[0053] Calculation of 50% Embryo Lethality (ELD) using the Reed and Muench method 50 The results showed that the virus had ELD. 50 According to the formula Log10ELD 50 = Logarithm of viral dilution above 50% + (Percentage above 50% - 50%) / (Percentage above 50% - Percentage below 50%) × Logarithm of dilution factor, used to determine ELD of DIBV2401 strain. 50 The value is 10 -4.47 / 0.2mL.

[0054] 5. Viral genome amplification and sequence analysis:

[0055] The genome of the isolated strain DIBVSD2401 was segmented and amplified, sequenced by Sangon Biotech (Shanghai, China), and the complete genome of the relevant avian coronavirus was retrieved from GenBank. Sequence alignment was performed using MAFFT. A maximum likelihood (ML) phylogenetic tree was constructed using MEGA and visualized using iTOL. Recombination analysis was performed using RDP v.4 software, and recombination events were validated using SimPlot (v3.5.1). The S protein sequence of the DIBVSD2401 strain was submitted to AlphaFold for structure prediction. The S protein structure was visualized using PyMOL (v2.3.3). The glycosylation sites of the S protein (N-glycosylation threshold > 0.5, O-glycosylation threshold > 0.1) were predicted using NetNGlyc and NetOGlyc tools.

[0056] The genome of strain DIBVSD2401 has a 5'UTR-1ab-S-3a-3b-EM-4b-4c-5a-5b-N-6b-3'UTR structure (see...) Figure 3 The nucleotide sequences of S, 3a, 3b, E, M, 4b, 4c, 5a, 5b, N, and 6b are shown in SEQ ID NO.1-SEQ ID NO.11, respectively.

[0057] Analysis of the S protein revealed multiple glycosylation sites, identifying 27 N-glycosylation sites, of which 70.4% were concentrated in the S1 region and 48% were located in the N-terminal receptor-binding domain (N-RBD). Simultaneously, 47 O-glycosylation sites were predicted, with 69.95% located in the S2 region. Structural prediction using AlphaFold showed that the S protein can form a trimer structure, and the receptor-binding sites are mainly concentrated at the N-terminus rather than the C-terminus. The N-RBD region contains a typical β-sheet structure. Furthermore, the two heptapeptide repeat regions (HR1 and HR2) of the S protein form a six-helix bundle, driving the fusion of the viral membrane with the host membrane (see...). Figure 3).

[0058] To further understand the genotype and genetic origin of strain DIBVSD2401, we compared it with other known IBV strains infecting birds. The results showed that DIBVSD2401 had high nucleotide similarity (87.0% to 96.3%) with chicken-derived IBV (CIBV) strains, while its similarity to duck coronavirus (DCoV) strain NC048214 was 77.9%, and it was even closer to the 2004 DCoVJF705860 strain (90.3%) (see...). Figure 4 Phylogenetic analysis showed that DIBVSD2401 closely clustered with chicken-derived IBV strains, while the two duck-derived IBV strains formed different phylogenetic groups (see...). Figure 5 ).

[0059] Further amino acid sequence analysis of the S protein homologs revealed that DIBVSD2401 shares 74.9%–98.3% amino acid identity with known CIBV strains; while its similarity to DCoV NC048214 is only 40.5%, and its similarity to the 2004 DCoV JF705860 strain is 92.1% (see [link to original text]). Figure 4 Phylogenetic analysis of the S protein was consistent with the phylogenetic results of the full genome sequence, indicating that DIBVSD2401 is most closely related to the CIBV strain (see...). Figure 5 ).

[0060] Because strain DIBVSD2401 showed high sequence similarity to CIBV strains, we further searched other CIBV genome sequences to determine the genotype of DIBVSD2401. The results showed that the whole-genome nucleotide similarity between DIBVSD2401 and GI-19 (QX subtype) ranged from 88.3% to 97.7% (see [link to relevant documentation]). Figure 6 Based on these sequence similarities, phylogenetic analysis further confirmed that DIBVSD2401 is most closely related to the GI-19 (QX subtype) strain, and the two cluster in the same clade (see...). Figure 7 Analysis of the S1 gene showed that DIBVSD2401 and GI-19 (QX subtype) exhibited 94.3%–98.8% similarity (see [link to relevant data]). Figure 6 Furthermore, the phylogenetic relationship of this gene is consistent with whole-genome analysis (see...). Figure 7 ).

[0061] In homology analysis, we noted a high degree of similarity between the ORF1ab region of DCoV NC048214 and DIBVSD2401, suggesting that DIBVSD2401 may have undergone gene recombination. Further recombination analysis confirmed that duck-derived IBV strain NC048214 is one of the potential parent strains of DIBVSD2401, and that DIBVSD2401 may have recombinated with multiple QX-like strains, ultimately forming the current DIBVSD2401 strain. Among the QX subtypes, KX252788 is the primary parent, but other QX subtype strains (such as KX252777) may also be potential parents (see [link to QX subtype analysis]). Figure 8 ).

[0062] Further sequence analysis revealed five insertion sequences from DCoV NC048214 in DIBVSD2401, located at positions 1060-1540, 4380-6860, 9160-11460, 20020-20340, and 26600-26700, respectively. The nucleotide sequences of these five sequences are shown in SEQ ID NO.12-SEQ ID NO.16, respectively. The first four sequences are primarily located in non-structural protein regions, accounting for 98.24% of the total recombinant sequences, while the fifth sequence is located in the structural protein N region (see...). Figure 9 ).

[0063] Based on the above results, strain DIBVSD2401 is named DIBVSD2401 Infectious Bronchitis Virus. This strain was deposited at the China Center for Type Culture Collection on December 26, 2024, with accession number CCTCC NO:V202503.

[0064] Example 2: Characterization of duck-derived recombinant infectious bronchitis virus DIBVSD2401

[0065] 1. Viral cell tropism:

[0066] To determine the cell tropism of duck-derived recombinant infectious bronchitis virus (DIBVSD2401), this invention examined the proliferation of DIBVSD2401 in different cell types, including chicken embryo fibroblasts (CEF) and duck embryo fibroblasts (DEF), as detailed below:

[0067] 1.1 Test Methods:

[0068] Primary duck embryo fibroblasts and chicken embryo fibroblasts were seeded in 24-well plates and cultured to 70% confluence. Cells were washed twice with PBS, infected with 1 MOI, and incubated at 37°C for 2 hours. Cells were washed three times with PBS to remove unadsorbed virus. Cells were maintained in DMEM with 2% FBS at 37°C and 5% CO2. Cells and culture supernatant were collected at different time points from 1 hour to 7 days, and viral RNA levels were detected by qPCR.

[0069] 1.2 Test Results:

[0070] Quantitative analysis of viral genomic RNA copy number using qRT-PCR showed that viral RNA levels in CEF reached approximately 5.15 × 10⁻⁶ after one day. 9 Copy / μg, then remain stable for 7 days (see Figure 10 Viral RNA levels in the DEF peaked at 5 days (2.59 × 10⁻⁶). 8 (copy / μg), then gradually decreased ( Figure 10 (Middle left figure). We also found that the viral load released from the CEF supernatant reached 3.02 × 10⁻⁶ at 5 days. 8 The number of copies / mL indicates that CEF is highly susceptible to DIBVSD2401. Figure 10 (Right image in the middle)

[0071] 2. Pathogenicity of the virus:

[0072] 2.1 Test Methods:

[0073] (1) Duckling infection experiment

[0074] Fifty one-day-old ducklings were randomly divided into a challenge group (n=25) and a control group (n=25). At five days of age, the challenge group was inoculated with 0.4 mL of ELD via nasal and ocular routes. 50 =10 -4.47 / 0.2mL) of viral fluid. The control group used an equal volume of PBS. Observations were conducted for 15 consecutive days, with samples collected periodically (3, 6, 9, 12, and 15 days post-infection). Samples were collected for RT-PCR, viral load testing, and histopathological analysis.

[0075] (2) Adult egg-laying duck infection experiment

[0076] Forty healthy 44-week-old laying ducks were divided into a challenge group (n=20) and a control group (n=20). The challenge group was inoculated with 0.4 mL of virus solution via nasal cavity and eye, while the control group received PBS. Clinical symptoms were monitored daily, and egg production was recorded. Fecal swabs and major organs were collected at 3, 5, 7, and 9 days post-infection (dpi) for RT-qPCR and pathological analysis.

[0077] 2.2 Test Results:

[0078] After ducklings were infected with DIBVSD2401, the infected ducklings did not show respiratory symptoms. However, by the 5th day of infection (dpi), the growth and development of the infected ducklings was significantly delayed (see...). Figure 11 By the 11th dpi, the growth retardation became more pronounced (see...). Figure 12 However, no deaths were observed within 15 days. The infected ducklings were found to shed the virus only through their digestive tract, and the virus was only detected in the kidneys and intestines (see...). Figure 13 No virus was detected in other organs or tissues, and no virus was detected in the blank control group either.

[0079] In laying ducks infected with DIBVSD2401, the egg production rate of the infected group decreased significantly. Simultaneously, a large number of thin-shelled, broken-shelled, sandy-shelled, and deformed eggs appeared (see...). Figure 14 The autopsy results showed follicular congestion, with newly formed follicles appearing reddish-brown or grayish-brown. Later follicles showed degeneration and deformation, and in severe cases, follicular liquefaction was observed (see...). Figure 15 The virus was detected in the oviducts, intestines, and kidneys of laying ducks after infection (see [link]). Figure 16 Furthermore, the egg production rate of ducks significantly decreased after infection (see...). Figure 17 ).

[0080] Example 3: Preparation and Performance Evaluation of Inactivated Vaccine

[0081] 1. Preparation of inactivated vaccines:

[0082] (1) Virus proliferation and harvest:

[0083] The duck-derived recombinant infectious bronchitis virus (DIBVSD2401) isolated in Example 1 was inoculated into chicken embryo fibroblasts (CEF) and incubated at 37°C for 2 hours. The cells were washed three times with PBS to remove unadsorbed virus. The cells were maintained in DMEM with 2% FBS and cultured at 37°C and 5% CO2 for 1 day. Cells were then disrupted by freeze-thaw cycles, and the supernatant was collected and purified to obtain the duck-derived recombinant infectious bronchitis virus solution.

[0084] (2) Inactivation of the virus fluid:

[0085] Virus solutions that pass bacterial testing are inactivated with formaldehyde. The inactivation conditions are: add formaldehyde to a final concentration of 0.2% and stir at 37°C for 16 hours.

[0086] (3) Vaccine preparation:

[0087] ① Preparation of the oil phase: Take No. 10 pharmaceutical white oil, Span-80 and aluminum stearate and mix them thoroughly in a volume ratio of 94:6:2. Sterilize by autoclaving at 121℃ for later use.

[0088] ② Preparation of the aqueous phase: Mix the completely inactivated virus solution with sterile Tween-80 at a volume ratio of 96:4 by shaking until the Tween-80 is completely dissolved.

[0089] ③ Emulsification: The oil phase and the water phase are mixed in a ratio of 2:1, and stirred continuously. After all the water phase is added, mix at 6000 rpm for 10 minutes, then emulsify at 8000 rpm for 20 minutes. Dispense into portions for use to prepare the inactivated vaccine.

[0090] 2. Quality inspection of inactivated vaccines:

[0091] The quality inspection of the prepared inactivated vaccine was conducted in accordance with the "Veterinary Pharmacopoeia of the People's Republic of China" (2020 edition).

[0092] The results show that the inactivated vaccine prepared by this invention complies with the relevant provisions of the "Veterinary Pharmacopoeia of the People's Republic of China" (2020 edition).

[0093] 3. Protective testing of inactivated vaccines

[0094] Thirty 5-day-old ducklings were randomly divided into three groups of 10 each. Group 1 was the immunization group, receiving an intramuscular injection of the inactivated vaccine prepared in this embodiment (0.5 mL / duckling) in the leg. Group 2 was the control group, receiving an intramuscular injection of an equal volume of sterile white oil adjuvant in the leg. Group 3 was the blank control group, receiving no treatment. One week after immunization, ducklings in groups 1 and 2 were intranasally inoculated with 0.6 mL of ELD (a type of inactivated vaccine). 50 =10 -4.47 / 0.2mL) of duck-derived recombinant infectious bronchitis virus (DIBVSD2401) was collected and isolated in groups. The respiratory symptoms, growth and development, and mortality of ducklings in each group were observed for 3 weeks.

[0095] The results showed that the immunized ducklings did not exhibit respiratory symptoms, and their growth and development were similar to those of the blank control group. No deaths occurred during the observation period. Compared with the blank control group, the ducklings in the control group showed stunted growth, and two ducklings died during the observation period. The results indicate that the inactivated vaccine prepared in this invention can provide effective protection for ducklings.

[0096] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A duck-derived recombinant infectious bronchitis virus strain, with accession number CCTCC NO:V202503.

2. The use of the duck-derived recombinant infectious bronchitis virus as described in claim 1 in the preparation of a vaccine for the prevention or treatment of avian infectious bronchitis.

3. The application according to claim 2, characterized in that, The avian infectious bronchitis was caused by a duck-derived recombinant infectious bronchitis virus with accession number CCTCC NO:V202503.

4. The application according to claim 2 or 3, characterized in that, The vaccine is either an inactivated vaccine or a live attenuated vaccine.

5. The application according to claim 4, characterized in that, The vaccine in question is an inactivated vaccine.

6. A vaccine for the prevention or treatment of avian infectious bronchitis, characterized in that, The vaccine contains an immunogenically effective amount of duck-derived recombinant infectious bronchitis virus fluid; the duck-derived recombinant infectious bronchitis virus has the preservation number CCTCC NO:V202503.

7. The vaccine according to claim 6, characterized in that, The viral fluid of duck-derived recombinant infectious bronchitis virus was prepared by the following method: The duck-derived recombinant infectious bronchitis virus with accession number CCTCC NO:V202503 was inoculated into duck embryo fibroblasts or chicken embryo fibroblasts. After the duck-derived recombinant infectious bronchitis virus was proliferated and cultured, the cells were broken by freeze-thaw, the supernatant was collected and purified to obtain the viral fluid of duck-derived recombinant infectious bronchitis virus.

8. The vaccine according to claim 6, characterized in that, The vaccine also contains adjuvants.

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