Avian coronavirus YB-WF strain and inactivated vaccine and application thereof

Abstract

The invention provides a novel avian coronavirus YB-WF strain as well as an inactivated vaccine and application thereof, and belongs to the technical field of poultry vaccine preparation. According to the invention, coronavirus nucleic acid is detected from multiple egg drop and diarrhea cases of laying hens, the pathogen is initially considered to be chicken IBV, but chicken IBV is not separated from SPF chick embryos, and then whole genome sequencing and phylogenetic analysis find that the avian IBV is not separated from the SPF chick embryos. The avian coronavirus disclosed by the invention is formed by recombining an S gene similar to a turkey coronavirus and a skeleton gene (other genes except the S gene, the same hereinafter) of a GI-19 pedigree infectious bronchitis virus. Transmission electron microscope observation, RT-PCR (Reverse Transcription-Polymerase Chain Reaction) test, gene sequencing and animal regression test show that the screened virus is an avian coronavirus strain, can proliferate in the intestinal tract of an SPF (Specific Pathogen Free) chick embryo and causes the clinical manifestation of the SPF chicken blue crown disease, which is of great significance for preventing and controlling the disease in laying hens in China.

Description

Technical Field

[0001] The present invention belongs to the technical field of poultry vaccine preparation, and particularly relates to an avian coronavirus strain YB-WF, an inactivated vaccine obtained therefrom, and its uses. Background Art

[0002] Among poultry, avian coronavirus - infectious bronchitis virus (hereinafter referred to as IBV) of the genus deltacoronavirus is the pathogen causing infectious bronchitis in chickens (hereinafter referred to as IB). Since it was first reported in 1931, it has been distributed worldwide and has become one of the important diseases affecting the poultry industry production.

[0003] Bluecomb, also known as Corcnaviral Enteritis, is an acute and highly contagious disease that can infect turkeys of all ages, characterized clinically by loss of appetite, constant chirping, weight loss, mental depression, diarrhea, and decreased egg production. Peterson and Hymas first reported this disease in 1951 and called it "swamp fever". In the following decades, this disease has broken out successively in global turkey farming areas, causing huge losses. However, so far, there has been no research and report on the disease caused by turkey bluecomb in chickens in China. Summary of the Invention

[0004] The present invention provides an avian coronavirus strain YB-WF, an inactivated vaccine obtained therefrom, and its uses. The YB-WF strain has been experimentally shown to be an avian coronavirus strain that can proliferate in the intestines of SPF chicken embryos and cause the clinical manifestations of bluecomb in SPF chickens, which is of great significance for preventing and controlling this disease in laying hen farms in China.

[0005] To achieve the above object, the present invention provides an avian coronavirus strain YB-WF, which was deposited at the China Center for Type Culture Collection in Wuhan on February 11, 2025, with the deposit number CCTCC NO: V202509.

[0006] Preferably, the avian coronavirus is recombinantly formed by sequentially connecting the first backbone gene of the GⅠ-19 lineage infectious bronchitis virus of chickens, the S gene of the turkey-like coronavirus, and the second backbone gene of the GⅠ-19 lineage infectious bronchitis virus of chickens.

[0007] Preferably, the nucleotide sequence of the S gene is as shown in SEQ ID NO: 1, the nucleotide sequence of the first backbone gene is as shown in SEQ ID NO: 2, and the nucleotide sequence of the second backbone gene is as shown in SEQ ID NO: 3.

[0008] The present invention also provides the use of the avian coronavirus YB-WF strain as an antigen according to the above technical solution in the preparation of an inactivated avian coronavirus vaccine.

[0009] The present invention also provides an inactivated avian coronavirus vaccine, which is prepared by using the avian coronavirus YB-WF strain according to the above technical solution.

[0010] Preferably, the preparation method is as follows:

[0011] Take 95 parts of veterinary white oil and 1 part of aluminum stearate, place them in an oil phase preparation tank, heat to 80 °C, add 5 parts of Span-80, when the temperature reaches 115 °C, maintain for 30 min, and cool for later use;

[0012] Mix 96 parts of the inactivated avian coronavirus YB-WF strain antigen solution with 4 parts of sterilized Tween-80, and stir in an aqueous phase preparation tank until Tween-80 is completely dissolved;

[0013] Take 2 parts of the oil phase and place it in a high-speed shearing machine, rotate and stir slowly, and at the same time slowly add 1 part of the aqueous phase, emulsify at 10000 r / min for 5 minutes. After emulsification, take 10 ml and centrifuge at 3000 r / min for 15 minutes to obtain the product.

[0014] Preferably, the content of the avian coronavirus YB-WF strain in the inactivated avian coronavirus vaccine is 10 -6.5 EID 50 / 0.2 mL. When the immunization dose is 1 mL, the efficacy of the inactivated avian coronavirus vaccine reaches 100% protection

[0015] The present invention also provides the use of the inactivated avian coronavirus vaccine according to any one of the above technical solutions in the challenge protection against the avian coronavirus YB-WF strain.

[0016] The present invention also provides the use of the inactivated avian coronavirus vaccine according to any one of the above technical solutions as a medicament for preventing and controlling blue comb disease caused by avian coronavirus.

[0017] Compared with the prior art, the advantages and positive effects of the present invention are as follows:

[0018] The nucleic acid of coronavirus has been detected in multiple cases of laying hens with decreased egg production and diarrhea. Initially, it was thought that the pathogen was chicken IBV, but chicken IBV was not isolated in SPF chicken embryos. Later, through whole-genome sequencing and phylogenetic analysis, it was found that the avian coronavirus in the present invention was recombined by the S gene of a turkey coronavirus-like and the backbone genes (other genes except the S gene, the same below) of the GI-19 lineage infectious bronchitis virus of chickens. Through transmission electron microscopy observation, RT-PCR test, gene sequencing and animal regression test, it was shown that the virus screened in the present invention was an avian coronavirus strain, which could proliferate in the intestines of SPF chicken embryos and cause the clinical manifestations of blue comb disease in SPF chickens, which was of great significance for the prevention and control of this disease in laying hen farms in China. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Transmission electron microscopy observation of avian coronavirus particles provided by the embodiment of the present invention (Bar = 100nm);

[0020] Figure 2 Phylogenetic analysis based on the whole genome of avian coronavirus provided by the embodiment of the present invention;

[0021] Figure 3 Phylogenetic analysis based on the backbone genes of avian coronavirus provided by the embodiment of the present invention;

[0022] Figure 4 Phylogenetic analysis based on the S gene of avian coronavirus provided by the embodiment of the present invention;

[0023] Figure 5 Fecal conditions of each group of SPF chickens on the 3rd day after virus challenge provided by the embodiment of the present invention, where a is group 1 (control group), and b is group 2 (virus challenge group);

[0024] Figure 6 Duodenal autopsy changes of each group of SPF chickens at different times after virus challenge provided by the embodiment of the present invention, where a, b, c, d, e are on the 3rd day after virus challenge; A, B, C, D, E, F are on the 5th day after virus challenge; a, b, A, B, C are group 1; c, d, e, D, E, F are group 2;

[0025] Figure 7 Histopathological changes 5 days after virus challenge provided by the embodiment of the present invention, where a, b are group 1 (control group); c, d are group 2 (virus challenge group);

[0026] Figure 8 Virus loads in different organs after virus challenge provided by the embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0027] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] Example 1 Isolation of YB-WF strain

[0029] From the end of 2018 to the beginning of 2024, from late autumn to early spring of the following year, laying hens sent for inspection in Henan, Hebei, Shandong, Guizhou and other places suddenly showed clinical symptoms of decreased egg production and diarrhea without any warning, and the diseased samples were collected and sent to the laboratory for testing.

[0030] The trachea, bursa of Fabricius and intestine of sick chickens were processed separately, 5 times the volume of PBS was added, shredded, fully homogenized and repeatedly frozen and thawed 3 times, centrifuged at 8000g for 15min, and the supernatant was filtered and sterilized with a 0.22μm filter. Scheme 1: 6.5-day-old SPF chicken embryos and 8-day-old turkey / guinea fowl embryos were inoculated through the yolk sac route, and 9.5-day-old and 15-day-old SPF chicken embryos and 12-day-old and 24-day-old turkey / guinea fowl embryos were inoculated through the allantoic cavity route, 0.2mL / piece, cultured in a 37℃ incubator, and the embryos were illuminated twice a day, and observed continuously until the chicken embryos pecking the shells. The pecking chicken embryos were frozen at 4℃ overnight, and the chicken embryo intestinal tissues were harvested, and the above method was used for 10 consecutive blind passages. Scheme 2: Inoculate the monolayer of Vero cells, LMH cells, CEF cells, DF-1 cells, and MA-104 cells that fill the cell bottle, adsorb at 37℃ for 1h with 5% inoculation, pour off the adsorption solution, add 5mL of maintenance solution containing 20μg / mL trypsin, and culture continuously at 37℃ for 5-7 days, and observe the cell status every day. When the cell solution turns yellow, freeze and thaw the cell bottle repeatedly at -20℃ for 3 times, centrifuge at 5000g, and take the supernatant to pass blindly for 5 generations in the same way as above.

[0031] After the chicken embryos were inoculated through the allantoic cavity route, the chicken embryos did not die and the embryos showed no lesions. After 10 consecutive generations of blind transmission, the embryos showed no "curling" phenomenon and could hatch normally. Nucleic acid tests found that the viruses could reproduce in the intestines of the embryos, but no viral nucleic acid was detected in the allantoic fluid, yolk, and embryo surface. The virus could not be isolated through the yolk sac route. The experiment found that the virus grew best in turkey embryos and guinea fowl embryos. After the samples were inoculated into cells, it was found that the viruses could not grow and reproduce in the above cell lines, and the nucleic acid test was negative.

[0032] The avian coronavirus successfully isolated via the allantoic cavity route was named YB-WF. It was deposited at the China Center for Type Culture Collection in Wuhan on February 11, 2025, with the deposit number CCTCC NO: V202509. The avian coronavirus was recombinantly formed by successively linking the first backbone gene of the GⅠ-19 lineage infectious bronchitis virus of chickens, the S gene of the turkey coronavirus-like, and the second backbone gene of the GⅠ-19 lineage infectious bronchitis virus of chickens. Among them, the nucleotide sequence of the S gene is as shown in SEQ ID NO: 1, the nucleotide sequence of the first backbone gene is as shown in SEQ ID NO: 2, and the nucleotide sequence of the second backbone gene is as shown in SEQ ID NO: 3.

[0033] Example 2 Identification of the YB-WF Strain

[0034] 2.1 Transmission Electron Microscopy Observation

[0035] Take the intestinal grinding fluid of the blindly passaged F5 generation embryos, add 100 ml of sterile physiological saline, centrifuge at 10,000×g for 20 min to remove larger impurities, and then centrifuge at 40,000×g for 3 h at 4°C to further remove smaller impurities. Resuspend the virus particles with 60 μl of PBS and negatively stain with 1% phosphotungstic acid for 30 s, and observe the virus particle structure under a transmission electron microscope.

[0036] As Figure 1 shown, the diameter of the virus particles in the electron microscope field of view is about 60 nm to 140 nm. The virus is spherical or oval, surrounded by an envelope, and the envelope is covered with spikes like a corona, which conforms to the structural characteristics of coronaviruses.

[0037] 2.2 Hemagglutination Identification

[0038] Take three samples of the allantoic fluid, yolk, and intestinal homogenate of the 3rd generation SPF chicken embryos. Scheme 1: Prepare 1% erythrocyte suspensions of chickens, rabbits, and mice by conventional methods and measure the hemagglutination activity according to the microhemagglutination test method. Scheme 2: Add 1% trypsin and incubate at 37°C for 3 h, and perform hemagglutination tests with the erythrocytes of chickens, rabbits, and mice.

[0039] The results showed that inoculation of the allantoic fluid, yolk, and intestinal homogenate of chicken embryos could not agglutinate the erythrocytes of chickens, rabbits, and mice. After treating the samples with 1% trypsin, they still could not agglutinate the erythrocytes of chickens, rabbits, and mice, and the control group samples also did not agglutinate the above erythrocytes.

[0040] 2.3 PCR Identification

[0041] Extract the RNA and DNA of the allantoic fluid of SPF chicken embryos using RNA and DNA extraction kits respectively, and perform nucleic acid detection on common avian disease pathogens such as avian reovirus, avian astrovirus, chicken parvovirus, chicken IBV, avian encephalomyelitis virus, avian adenovirus, and avian nephritis virus (Table 1) by PCR or RT-PCR. RT-PCR reaction system (25 μL): PrimeScript 1step Enzyme Mix 1.0 μL, 2×1step Buffer 12.5 μL, upstream and downstream primers (20 μmol / L) 0.5 μL each, RNA template 5 μL, supplemented with ddH2O to 25 μL. RT-PCR reaction conditions: reverse transcription at 50 °C for 30 min; pre-denaturation at 95 °C for 5 min; 95 °C for 30 s, 55 °C for 30 s, 72 °C for 60 s, 35 cycles; extension at 72 °C for 5 min, stored at 12 °C; PCR reaction system (20 μL): PremixTaq 10 μL, upstream and downstream primers (20 μmol / L) 0.5 μL each, DNA template 2 μL, ddH2O 7 μL. PCR reaction conditions: pre-denaturation at 95 °C for 5 min; 95 °C for 30 s, 55 °C for 30 s, 72 °C for 60 s, 35 cycles; extension at 72 °C for 5 min, stored at 12 °C. The RT-PCR and PCR products were detected by 1.0% agarose gel electrophoresis and photographed.

[0042] Table 1: Primer sequences of common viruses

[0043]

[0044]

[0045] Note: F represents the upstream primer; R represents the downstream primer

[0046] After the PCR reaction, the products were detected by 1.0% agarose gel electrophoresis to analyze the amplification results and photographed. The electrophoresis results showed that only the IBV nucleic acid in the ground chicken embryo intestine was positive, and no suspected pathogens were detected in other tissue and cell samples.

[0047] Thirteen collected diseased samples were isolated and detected for pathogens one by one. The results showed that all samples were positive for avian coronavirus (Table 2).

[0048] Table 2 Statistical situation of pathogen isolation and detection of 13 samples

[0049]

[0050] Note: AvCoV is avian coronavirus

[0051] Example 3 Genome amplification and sequencing analysis

[0052] The whole genome sequence of the isolated avian coronavirus was determined by next-generation sequencing technology, and finally the full-length avian coronavirus genome was obtained. The sequences of known avian coronavirus (avian infectious bronchitis virus and turkey coronavirus) isolates were downloaded from GenBank. Using the DNAMAN software, the positions and numbers of ORFs of each gene segment were predicted, the amino acid sequences were deduced, and the gene coding conditions were analyzed by BLASTn. The nucleotide sequence homology of different chicken-derived strains was compared using ClustalW1.8, and a phylogenetic tree was constructed through MEGA6.0. The nucleotide homology of the measured avian coronavirus YB-WF strain was compared with other avian coronavirus sequences published on GenBank.

[0053] Table 3 Comparison of gene homology (%) between avian coronavirus YB-WF strain and chicken IBV and turkey coronavirus

[0054]

[0055] As shown in Table 3, the nucleotide homologies of the avian coronavirus in this study with the chicken IBV genome, S gene, and backbone gene were 83.0% - 90.1%, 51.4% - 52.1%, and 87.1% - 95.2% respectively, and the nucleotide homologies with the turkey / guinea fowl coronavirus genome, S gene, and backbone gene were 83.9% - 84.0%, 76.7% - 77.5% / 86.7% - 87.1% respectively. This indicates that the isolated avian coronavirus strain in this study is different from both chicken IBV and turkey coronavirus and is a type of avian coronavirus.

[0056] The above avian coronavirus YB-WF strain was compared and analyzed with the full genome sequences, backbone gene sequences, and S gene sequences of chicken IBV and turkey / guinea fowl coronavirus published on GenBank, and a phylogenetic tree was drawn ( Figures 2 to 4 ). The results showed that although the S gene sequence of the avian coronavirus isolated in this study was relatively closely related to the S gene of the turkey / guinea fowl coronavirus prevalent in Europe and America at the end of the last century, it was not in the same evolutionary branch and had mutated; the backbone gene was in the same evolutionary branch as the GⅠ-19 lineage avian infectious bronchitis virus prevalent in China in recent years; in summary, the evolution of the backbone gene and S gene indicates that the avian coronavirus in this study may be related to the recombination of the GⅠ-19 lineage avian infectious bronchitis virus starting from China and the turkey / guinea fowl coronavirus prevalent in Europe and America, and it is a recombinant virus.

[0057] According to the division by the whole genome or the spike S gene, the novel coronaviruses isolated from poultry at present in this study were divided into three categories: Category Ⅰ is chicken IBV; Category Ⅱ is turkey / guinea fowl coronavirus; Category Ⅲ is recombinant avian coronavirus.

[0058] Example 4 Regression Test

[0059] The avian coronavirus strain YB-WF isolated in this study was inoculated into 21-day-old SPF chickens. The results showed that on the 2nd to 3rd day after inoculation, the chickens in Group 2 (the inoculated group) showed rapid diarrhea, with a lot of fecal droppings and watery feces, and the feces were not formed, which was significantly different from that of the chickens in Group 1 (the control group) (the feces were dry and formed). Figure 5 ) As time went by, after 7 to 10 days, the diarrhea in the chickens of Group 2 gradually stopped, and the whole course of the disease was relatively short. During the experiment, individual chickens in the inoculated group only showed a short-term listless phenomenon, but there was no death. On the 3rd day and the 5th day after inoculation, 3 chickens from each of the two groups were sacrificed respectively, and it was found that the main lesions were in the intestine, with the duodenum being the most serious. The intestines of the chickens in Group 2 were dilated, flaccid, pale, the intestinal mucosa fell off, and there was incompletely digested chyme inside. Most chickens had intestinal bleeding, and the pancreas was swollen and pale. Figure 6 )

[0060] On the 5th day after inoculation, after sacrificing the SPF chickens in Group 2, a little shedding of the duodenal villi was observed, with mild congestion and bleeding at the top of the villi, and congestion and bleeding in the lamina propria of the mucosa. Figure 7 d); Granular degeneration of the renal tubular epithelial cells and interstitial congestion Figure 7 e); The bursa of Fabricius lymphoid follicles developed well, and no abnormal lesions were found Figure 7 f). There were no obvious histological changes in the control group Figure 7 a-c).

[0061] Three chickens were sacrificed respectively at 3 dpi, 5 dpi, 7 dpi, 9 dpi, 14 dpi, and 21 dpi, and the virus load was measured in each organ. The fluorescence quantitative PCR data showed that the virus load was relatively high in the bursa of Fabricius, cecal tonsils and intestinal system. The virus load in each organ remained at a relatively high level in the first 7 days after inoculation, and then gradually decreased. The pathogen could hardly be detected in some organs at 21 dpi. The organs that could still detect the pathogen at 21 dpi were the liver, bursa of Fabricius, jejunum, ileum, cecum and rectum. Figure 8 )

[0062] Through the above electron microscopy observation, hemagglutination experiment, gene sequencing analysis, and animal regression experiment, it was judged according to Koch's postulates that the pathogen causing egg production decline and diarrhea in laying hens clinically was avian coronavirus. The establishment of this "animal model" laid a foundation for the subsequent development of our vaccines.

[0063] Example 5 Preparation of Vaccine and Virus Neutralization Test

[0064] 5.1 Preparation of Vaccine

[0065] 5.1.1 Propagation of Avian Coronavirus

[0066] The avian coronavirus YB-WF strain (the YB-WF strain is an SPF chicken embryo-adapted strain, isolated, identified and preserved by Qingdao Yibang Bioengineering Co., Ltd.) was inoculated into 15-day-old SPF chicken embryos, continuously observed for 5 to 6 days to propagate the required antigen, candled once a day, and the chicken embryos that died within 48 hours were discarded. The chicken embryos from 48 hours later to hatching were collected and cooled at 2 to 8 °C for 4 to 24 hours.

[0067] 5.1.2 Harvest

[0068] Take out the cooled chicken embryos, harvest the intestinal tissues of the chicken embryos, cut the intestinal tissues into pieces with scissors and grind them thoroughly (add quartz sand), then transfer the grinding liquid to a sterilized bottle, freeze-thaw it three times in a -80 °C refrigerator, centrifuge at 5000 rpm for 30 min in a 4 °C centrifuge, and carefully transfer the supernatant to a sterile bottle. Conduct sterility tests on the above venom according to the current "Chinese Veterinary Pharmacopoeia". The harvested supernatant was stored at 2 to 8 °C before inactivation.

[0069] 5.1.3 Inactivation

[0070] Add 10% formaldehyde solution to the harvested avian coronavirus liquid to make the final concentration of formaldehyde 0.1%. After shaking and inactivating at 200 rpm in a 37 °C shaker for 24 hours, take it out and store it at 2 to 8 °C.

[0071] 5.1.4 Semi-finished product inspection

[0072] (1) Sterility test

[0073] Conduct sterility tests according to the appendix of the current "Chinese Veterinary Pharmacopoeia" to determine sterility contamination.

[0074] (2) Virus content determination

[0075] The harvested virus liquid (sampled before inactivation), the YB-WF strain, was serially diluted 10-fold with sterilized normal saline, and 10 -4 、10 -5 、10 -6 、10 -7 4 dilutions were taken and inoculated into 15-day-old SPF chicken embryos via the allantoic cavity route, 0.2 ml per embryo. At the same time, 5 controls inoculated with normal saline were set, 0.2 ml per embryo. Incubate at 37 °C, candled twice a day, and observe until pecking the shell. Calculate EID 50 , the virus content is not less than 10 6.5 EID 50 / 0.2 ml.

[0076] (3) Inactivation test

[0077] Six 15-day-old SPF chicken embryos were taken, and 0.2 ml of inactivated virus solution was inoculated into the allantoic cavity of each embryo. The embryos were candled twice a day for 7 days. Meanwhile, the intestines of the chicken embryos were collected and blindly passaged twice according to the above method, and the death of the chicken embryos was observed and recorded. The results showed that the inactivation was complete and there were no dead embryos.

[0078] 5.1.5 Preparation of Inactivated Vaccine

[0079] The semi-finished antigen that passed the inspection was used for vaccine preparation (the volume ratio of each liquid component in the following preparation is calculated).

[0080] (1) Preparation of oil phase

[0081] Take 95 parts of veterinary white oil and 1 part of aluminum stearate, place them in the oil phase preparation tank and heat to 80 °C, then add 5 parts of Span-80. When the temperature reaches 115 °C, maintain for 30 minutes, and cool for later use.

[0082] (2) Preparation of water phase

[0083] Mix 96 parts of the inactivated avian coronavirus YB-WF strain antigen solution with 4 parts of sterilized Tween-80, and stir in the water phase preparation tank until Tween-80 is completely dissolved.

[0084] (3) Emulsification

[0085] Take 2 parts of the oil phase and place it in a high-speed shear mixer. Start the motor and stir slowly. At the same time, slowly add 1 part of the water phase and emulsify at 10,000 r / min for 5 minutes. After emulsification, take 10 ml and centrifuge at 3,000 r / min for 15 minutes. The water phase separated at the bottom of the tube should not exceed 0.5 ml.

[0086] 5.1.6 Inspection of Vaccine Finished Product

[0087] 5.1.6.1 Characteristics

[0088] Appearance: The vaccine is a milky white emulsion;

[0089] Dosage form: It is a water-in-oil type. Take a clean pipette, suck a small amount of the vaccine and drop it into cold water. Except for the first drop, it does not spread.

[0090] Stability: Suck 10 ml of the vaccine into a centrifuge tube and centrifuge at 3,000 r / min for 15 minutes. The water phase separated at the bottom of the tube should exceed 0.5 ml.

[0091] Viscosity: It is carried out according to the appendix of the current "Chinese Veterinary Pharmacopoeia" and meets the requirements.

[0092] 5.1.6.2 Safety Inspection

[0093] Ten 7-day-old SPF chickens were used. Each chicken was subcutaneously injected with 1.0 ml of the vaccine at the neck. At the same time, five control chickens were set up respectively and raised under the same conditions. They were continuously observed for 14 days, and the food intake, water intake and clinical conditions of the experimental chickens were recorded. No local or systemic adverse reactions caused by the vaccine occurred.

[0094] The results showed that the chickens in the experimental group and the control group developed normally and had good mental states. After autopsy of the experimental group, it was found that the vaccine was well absorbed at the injection site, and there were no inflammatory reactions such as swelling, redness or tissue necrosis. This indicated that the trial-produced vaccine was safe and harmless and had no impact on the growth of animals.

[0095] 5.1.6.3 Potency test

[0096] Ten 21-day-old SPF chickens were used. Each chicken was subcutaneously injected with avian coronavirus vaccine at the neck, 0.2 ml per chicken. Another five chickens of the same age were not immunized as controls. Twenty-one days after immunization, blood was collected to separate serum, and the antibodies were measured respectively. The antibody titer of the immunized group was not lower than 1200. The control group should all be negative.

[0097] 5.2 Virus cross-neutralization test

[0098] The avian coronavirus YB-WF strain, chicken infectious bronchitis virus H120 strain and turkey coronavirus TCoV-F strain were made into inactivated vaccines by culturing in SPF chicken embryos according to the above method. Twenty-one-day-old SPF chickens were immunized respectively, and the immunization dose was 0.5 ml per chicken. Immunization was carried out once every two weeks for a total of three times. Two weeks after the third immunization, blood was collected to separate serum. The venom of the YB-WF strain, H120 strain and TCoV-F strain was diluted to 200 EID 50 / 0.2 ml. The virus and serum were mixed in equal volume pairwise. After shaking and neutralizing at 37°C for 1 hour, five 15-day-old SPF chicken embryos were inoculated with each neutralization group of the YB-WF strain, H120 strain and TCoV-F strain, 0.2 ml per embryo. The embryo inoculation situation was observed daily until hatching.

[0099] Table 4 Results of serum cross-neutralization test of avian coronavirus YB-WF3 strain, chicken infectious bronchitis virus H120 strain and turkey coronavirus TCoV-F strain

[0100]

[0101] Note: * indicates virus; ** indicates serum. When the R value is greater than 0.8, it is the same serotype. When the R value is between 0.25 and 0.8, it is different subtypes of the same serotype. When the R value is less than 0.25, it is a different serotype.

[0102] As shown in Table 4, the neutralization ability of the positive sera of the YB-WF strain, H120 strain and TCoV-F strain with their own homologous positive sera was relatively strong, but the neutralization ability against each other was relatively poor, and the three belonged to different serotypes.

[0103] Example 6 Virulence Challenge Protection Test

[0104] The avian coronavirus strain YB-WF, the infectious bronchitis virus strain H120 of chickens, and the turkey coronavirus strain TCoV-F were inoculated into the allantoic cavity of chicken embryos for cultivation, and the EID50 was measured. Antigens were prepared using the F8-generation venom after passage culture. The antigen titers of the virus strains were 10 -6.5 EID 50 / 0.2 mL. They were inactivated with formaldehyde at a final concentration of 1.0‰ at 37°C for 24 h, and inactivated vaccines were prepared according to the ratio of oil phase: water phase = 2:1. Seventy 7-day-old SPF chickens were divided into 7 groups, with 10 chickens in each group. The chickens in the immunized groups were subcutaneously injected with the inactivated YB-WF avian coronavirus vaccine in the neck, 0.5 ml per chicken, and a challenge control group (not immunized, directly challenged) and a blank control group (not immunized, not challenged) were set up. The second immunization was carried out at 21 days of age. Fourteen days after the second immunization, the chickens were orally challenged with the avian coronavirus strain YB-WF, 0.5 ml per chicken (YB-WF = 10 -3.82 EID 50 / 0.1 mL). The mental state, food intake, and disease occurrence of the chicken flock were observed daily. Cloacal swabs were collected from each chicken on the 2nd, 4th, 6th, 8th, and 10th days after challenge, and the virus excretion was detected by RT-PCR, and the experimental data were recorded (Table 5).

[0105] Table 5 Virus Excretion after Coronavirus Challenge

[0106]

[0107] The results of the virulence challenge protection test showed that the chickens in the YB-WF immunized group did not excrete the virus and had no abnormal clinical manifestations such as diarrhea after being challenged with this virus, but when challenged with the H120 and TCoV viruses, the chicken flock showed virus excretion; the chicken flocks in the H120 immunized group and the TCoV immunized group could play a good protective role when challenged with this virus, but after being challenged with the YB-WF strain, different degrees of virus excretion occurred, and some chickens also had diarrhea and reduced food intake. The SPF chickens in the challenge control group excreted the virus and had diarrhea, and the SPF chickens in the blank control group were normal, indicating that the experiment was valid. The above experimental data showed that the inactivated vaccines prepared from the above three virus strains could only play a good protective role against this virus, and the cross-protective effect among the three was very poor. To sum up, the content of the avian coronavirus strain YB-WF in the inactivated avian coronavirus vaccine developed in this study was 10 -6.5 EID 50 / 0.2 mL. When the immunization dose was 1 mL, the inactivated avian coronavirus vaccine achieved 100% protection against this virus, and the other two could not effectively protect against the attack of the YB-WF strain on SPF chickens, and the immune efficacy of the vaccine was poor.

[0108] It can also be seen from Table 5 that the serum cross-protection between avian coronavirus strain YB-WF and infectious bronchitis virus strain H120 of chickens and turkey coronavirus strain TCoV-F is poor, and they do not belong to the same serotype. Given that avian coronavirus strain YB-WF has started to prevail in some laying hen breeding areas in China at present, using this strain to prepare a vaccine can play a very good preventive role in clinical diseases such as diarrhea and decreased egg production caused by this prevalent virus.

Claims

1. Avian coronavirus YB-WF strain, characterized in that It was deposited in the China Center for Type Culture Collection in Wuhan on February 11, 2025, with the deposit number being CCTCC NO: V202509.

2. The avian coronavirus YB-WF strain according to claim 1, characterized in that Avian coronavirus is composed of the first backbone gene of the GⅠ-19 lineage infectious bronchitis virus, the S gene of the turkey coronavirus, and the second backbone gene of the GⅠ-19 lineage infectious bronchitis virus.

3. The avian coronavirus YB-WF strain according to claim 2, characterized in that The nucleotide sequence of the S gene is shown in SEQ ID NO: 1, the nucleotide sequence of the first backbone gene is shown in SEQ ID NO: 2, and the nucleotide sequence of the second backbone gene is shown in SEQ ID NO:

3.

4. Use of the avian coronavirus YB-WF strain according to claim 1 as an antigen in the preparation of an avian coronavirus inactivated vaccine.

5. An inactivated avian coronavirus vaccine, characterized in that: It is prepared using the avian coronavirus YB-WF strain described in claim 1.

6. The avian coronavirus inactivated vaccine according to claim 5, characterized in that The preparation method is as follows: Take 95 parts of veterinary white oil and 1 part of aluminum stearate, place them in an oil phase preparation tank and heat to 80°C, then add 5 parts of Siben-80, and maintain the temperature for 30 minutes when it reaches 115°C, and then cool it for use; 96 parts of the inactivated avian coronavirus YB-WF strain antigen liquid were mixed with 4 parts of sterilized Tween-80, and stirred in an aqueous phase preparation tank until the Tween-80 was completely dissolved; Take 2 portions of the oil phase and put them in a high-speed shearing machine, stir slowly, and slowly add 1 portion of the water phase, emulsify at 10,000 r / min for 5 minutes. After emulsification, take 10 ml and centrifuge at 3,000 r / min for 15 minutes.

7. The avian coronavirus inactivated vaccine according to claim 5, characterized in that The content of avian coronavirus YB-WF strain in the avian coronavirus inactivated vaccine is 10 -6.5 EID 50 / 0.2mL, when the immunization dose is 1mL, the efficacy of the inactivated avian coronavirus vaccine reaches 100% protection.

8. Use of the inactivated avian coronavirus vaccine according to any one of claims 5 to 7 in the protection against attack of avian coronavirus YB-WF strain.

9. Use of the inactivated avian coronavirus vaccine according to any one of claims 5 to 7 in a medicament for preventing and controlling blue crown disease caused by avian coronavirus.

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