Inactivated vaccine and egg yolk antibody for preventing and controlling goose astrovirus as well as preparation method and application of inactivated vaccine and egg yolk antibody

Abstract

The invention belongs to the technical field of veterinary biological products, and discloses an inactivated vaccine and egg yolk antibody for preventing and controlling goose astrovirus as well as a preparation method and application of the inactivated vaccine and egg yolk antibody. The invention discloses a goose astrovirus named as N-GoAstV-hs2403, the inventor obtains the goose astrovirus N-GoAstV-hs2403 with excellent immunogenicity through long-term research and separation, high-titer goose astrovirus liquid is prepared on the basis of the goose astrovirus N-GoAstV-hs2403, the titer is FAID50 = 108.50 / mL, an efficient inactivated vaccine and an egg yolk antibody are further developed, and the goose astrovirus N-GoAstV-hs2403 can be used for preparing the goose astrovirus. The titer and the production efficiency of the antibody are remarkably improved, and an innovative solution is provided for prevention and control of the goose astrovirus disease.

Description

Technical Field

[0001] This invention belongs to the field of veterinary biological products technology, specifically relating to an inactivated vaccine and egg yolk antibody for the prevention and control of goose astrovirus, as well as their preparation method and application. Background Technology

[0002] Goose astrovirus ( Goose Astrovirus Goose Astrovirus (N-GoAstV) is a single-stranded positive-sense RNA virus belonging to the genus *Avian Astrovirus* of the family Astroviridae. It is approximately 28-30 nm in diameter and lacks an envelope. It is a highly lethal infectious disease characterized by visceral urate deposition, primarily affecting goslings aged 2-25 days. Morbidity rates reach 50%-100%, with mortality rates reaching 30%-50%. Surviving geese experience growth retardation, causing significant economic losses to the goose farming industry. Etiological studies have confirmed that this disease is caused by a novel goose astrovirus (N-GoAstV), which differs significantly from classic strains in serological and biological characteristics.

[0003] At present, the prevention and control of goose astrovirus disease faces the following technical bottlenecks: (1) It is difficult for the virus to proliferate in vitro, and the low viral titer of traditional cell culture is difficult to meet the needs of vaccine production; (2) There is a lack of commercial vaccines and specific treatment drugs, and clinical treatment mainly relies on antibiotics for symptomatic treatment, which is not effective; (3) The existing egg yolk antibody preparation process uses antigens with insufficient immunogenicity, and the titer of hyperimmune egg antibodies is generally low, resulting in high production costs.

[0004] Therefore, it is urgent to develop a highly efficient and stable process to improve the proliferation efficiency of goose stellate virus and to develop a goose stellate virus yolk antibody with high neutralizing titer and protection rate that is easy to mass-produce. Summary of the Invention

[0005] The first objective of this invention is to provide a goose star virus.

[0006] The second objective of this invention is to provide the use of the goose astrovirus of the first aspect of this invention in the preparation of products for the prevention and control of diseases caused by goose astrovirus.

[0007] A third aspect of the present invention is to provide a vaccine.

[0008] The fourth aspect of this invention aims to provide a method for preparing the vaccine according to the fourth aspect of this invention.

[0009] The fifth aspect of this invention is to provide a goose stellate virus yolk antibody.

[0010] The sixth aspect of this invention aims to provide a method for propagating goose astrovirus.

[0011] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, a goose star virus strain, named N-GoAstV-hs2403, is provided and deposited at the China Center for Type Culture Collection on August 22, 2025, with accession number CCTCC NO:V202561.

[0012] The strain was isolated from goslings with clinical gout and was identified as a highly pathogenic goose astrovirus variant through gene sequencing and animal reinfection experiments.

[0013] A second aspect of the present invention provides the use of the goose astrovirus of the first aspect of the present invention in the preparation of products for the prevention and control of diseases caused by goose astrovirus.

[0014] In some embodiments of the present invention, the product includes reagents, drugs, and / or egg yolk antibodies.

[0015] In some embodiments of the present invention, the reagent includes diagnostic reagents.

[0016] In some embodiments of the present invention, the diagnostic reagent includes an antigen for diagnosing goose astrovirus. This antigen is an inactivated goose astrovirus of the first aspect of the present invention.

[0017] In some embodiments of the present invention, the drug includes a vaccine.

[0018] In some embodiments of the present invention, the vaccine includes an inactivated vaccine.

[0019] In some embodiments of the invention, the disease includes visceral gout in goslings caused by goose astrovirus infection.

[0020] A third aspect of the present invention provides a vaccine, the active ingredient of which comprises an inactivated virus of the goose astrovirus of the first aspect of the present invention.

[0021] In some embodiments of the present invention, the vaccine further includes pharmaceutically acceptable excipients.

[0022] In some embodiments of the present invention, the excipients include at least one of a carrier, a protective agent, an adjuvant, an excipient, or a diluent.

[0023] In some embodiments of the present invention, the excipients include Tween, white oil, and Span.

[0024] In some embodiments of the present invention, the immunization method of the vaccine includes nasal drops, injection, oral administration, soaking, or eye drops; preferably, it is administered by injection, such as intramuscular injection.

[0025] A fourth aspect of the present invention provides a method for preparing the vaccine according to the third aspect of the present invention, comprising the following steps: The goose astrovirus of the first aspect of the present invention is cultured and proliferated in goose retinal epithelial cells to obtain a viral solution; or, the goose astrovirus is inoculated into SPF goose embryos, incubated, and the allantoic fluid of the surviving embryos is collected to obtain a viral solution. Inactivation yields an inactivated virus solution; The inactivated virus solution was mixed with Tween to obtain an aqueous phase; The white oil and Span were mixed to obtain the oil phase; The oil and water phases are mixed and emulsified to obtain the vaccine.

[0026] In some embodiments of the present invention, the culture and proliferation includes the following steps: inoculating goose astrovirus into goose retinal epithelial cells at an MOI of 0.005-0.04, adding TPCK trypsin to the culture system, and adsorbing it in a cell culture incubator; supplementing cell maintenance medium and culturing for 10-20 hours; adding sodium butyrate to the culture system and culturing for 70-90 hours to obtain the virus solution.

[0027] In some embodiments of the present invention, goose astrovirus is inoculated into goose retinal epithelial cells at an MOI of 0.005-0.04; further inoculated at an MOI of 0.01-0.04; even further inoculated at an MOI of 0.01-0.02; and most preferably inoculated at an MOI of 0.01.

[0028] In some embodiments of the present invention, the final concentration of the TPCK trypsin in the culture system is 5-15 μg / mL; further, 8-15 μg / mL; even further, 10-12 μg / mL; and most preferably, 10 μg / mL.

[0029] In some embodiments of the present invention, the final concentration of sodium butyrate in the culture system is 1-15 mmol / L; further, 1-10 mmol / L; even further, 2.5-5 mmol / L; and most preferably 2.5 mmol / L.

[0030] In some embodiments of the present invention, the adsorption time is 0.5-6 h; further, 0.5-4 h; even further, 1-2 h; and most preferably 2 h.

[0031] In some embodiments of the present invention, when the goose stellate virus is inoculated into SPF goose embryos, the ELD of the goose stellate virus... 50 (1-9)×10 5.0 / mL; further, (3-6)×10 5.0 / mL.

[0032] In some embodiments of the present invention, the cell maintenance medium is serum-free DMEM-F12 medium.

[0033] In some embodiments of the present invention, the incubation conditions are 65-80 hours; more specifically, 70-75 hours.

[0034] In some embodiments of the present invention, the inactivation includes adding formaldehyde to the virus solution and inactivating it at 35-38°C for 20-30 hours. The final concentration of formaldehyde is 0.1%-0.3%.

[0035] In some embodiments of the present invention, the inactivated virus solution is mixed with Tween at a mass ratio of (35-40):1.

[0036] In some embodiments of the invention, the white oil and Span are mixed in a mass ratio of (15-19):1.

[0037] In some embodiments of the present invention, the volume ratio of the aqueous phase to the oil phase is 1:(1-2).

[0038] In a fifth aspect, the present invention provides a goose astrovirus yolk antibody obtained by immunizing laying hens with the vaccine of the third aspect of the present invention.

[0039] In some embodiments of the present invention, the goose astrovirus yolk antibody is prepared by a method comprising the following steps: immunizing laying hens with the vaccine; collecting hyperimmune eggs and separating the yolks; and extracting the yolks by acidification and precipitation with ammonium sulfate to obtain the goose astrovirus yolk antibody.

[0040] In some embodiments of the present invention, the acidification extraction is performed by mixing egg yolk with an acetate buffer (pH 4.5-5.5) at a volume ratio of 1:(7-9).

[0041] In some embodiments of the present invention, the ammonium sulfate precipitation includes graded precipitation using ammonium sulfate of different concentrations.

[0042] In some embodiments of the present invention, the fractional precipitation includes mixing the supernatant after acidification extraction with ammonium sulfate, wherein the ammonium sulfate saturation in the system is 45%-50%, and precipitating; taking the precipitate, redissolving it, and mixing it with ammonium sulfate to form a system with ammonium sulfate saturation of 60%-70%, and precipitating again.

[0043] This invention improves the recovery rate of the purification process combining acid extraction with fractional ammonium sulfate precipitation to over 80%, which is much higher than the traditional PEG precipitation method (approximately 60%).

[0044] In some embodiments of the present invention, the level of immunization of laying hens with the vaccine is as follows: Basic immunization: Subcutaneous injection of 1.0 mL of inactivated vaccine per bird; Second immunization: 14 days apart, 1.5 mL / bird; Third vaccination: 14 days later, 1.5 mL / bird; Maintain immunity: When the amplified titer of egg yolk agar reaches 1:96, administer a booster immunization of 1.5 mL / bird.

[0045] The immunization procedure provided by this invention enables the amplification titer of egg yolk agar to reach 1:1024, which is 4 times higher than that of conventional methods.

[0046] In some embodiments of the present invention, the method further includes steps such as dialysis, sterilization, concentration, and inactivation.

[0047] In some embodiments of the present invention, a protective agent may be added to the goose stellate virus yolk antibody as needed, and then freeze-dried to obtain a freeze-dried powder of goose stellate virus yolk antibody.

[0048] In some embodiments of the present invention, the protective agent includes trehalose, sucrose, mannitol, glycine, and arginine.

[0049] The protective agent of this invention ensures that the titer of freeze-dried egg yolk antibody products decreases by no more than 10% after 12 months of storage at 4°C, and the activity remains above 90% after 3 months of storage at room temperature (25°C), greatly improving the commercial applicability of the product.

[0050] A sixth aspect of the present invention provides a method for propagating goose astrovirus, comprising inoculating goose retinal epithelial cells with the goose astrovirus of the first aspect of the present invention, adding TPCK trypsin to the culture system and adsorbing it in a cell culture incubator; supplementing cell maintenance medium and culturing for the first time; adding sodium butyrate to the culture system and culturing for the second time.

[0051] This invention is the first to discover that goose stellate virus has high sensitivity in retinal epithelial cells, and that the goose stellate virus prepared in retinal epithelial cells by adding sodium butyrate to increase the viral titer has a higher titer than other cell lines.

[0052] In some embodiments of the present invention, goose astrovirus is inoculated into goose retinal epithelial cells at an MOI of 0.01-0.04; further inoculated at an MOI of 0.01-0.04; even further inoculated at an MOI of 0.01-0.02; and most preferably inoculated at an MOI of 0.01.

[0053] In some embodiments of the present invention, the final concentration of the TPCK trypsin in the culture system is 5-15 μg / mL; further, 8-15 μg / mL; even further, 10-12 μg / mL; and most preferably, 10 μg / mL.

[0054] In some embodiments of the present invention, the final concentration of sodium butyrate in the culture system is 1-15 mmol / L; further, 1-10 mmol / L; even further, 2.5-5 mmol / L; and most preferably 2.5 mmol / L.

[0055] In some embodiments of the present invention, the adsorption time is 0.5-6 h; further, 0.5-4 h; even further, 1-2 h; and most preferably 2 h.

[0056] In some embodiments of the present invention, the first culture time is 10-20 hours; more specifically, it is 12-18 hours.

[0057] In some embodiments of the present invention, the second culture time is 70-90 hours; more specifically, it is 80-90 hours.

[0058] In some embodiments of the present invention, the cell maintenance medium is serum-free DMEM-F12 medium.

[0059] The beneficial effects of this invention are: Through long-term research, the inventors isolated a goose astrovirus strain N-GoAstV-hs2403 with excellent immunogenicity, and prepared a high-titer goose astrovirus solution based on this strain, with a titer of FAID. 50 =10 8.50 / mL, and further developed highly efficient inactivated vaccines and egg yolk antibodies, significantly improving antibody titer and production efficiency, providing an innovative solution for the prevention and control of goose astrovirus disease.

[0060] The goose astrovirus provided by this invention was isolated from geese exhibiting typical visceral gout clinically, and confirmed as a variant strain by PCR. Animal reinfection experiments showed a 20% mortality rate in 3-day-old SPF goslings, and necropsy revealed urate deposits in the viscera. N-GoAstV-hs2403 exhibits strong immunogenicity, and the inactivated vaccine prepared from it shows high neutralizing antibody titers after immunization, with a challenge protection rate of 100%, significantly superior to existing strains.

[0061] The vaccine provided by this invention exhibits good stability and a long duration of antibody production after immunization; the antibody titer remains above 1:128 even 5 months after three immunizations. This vaccine provides 100% protection against visceral gout in goslings, offering a safe and efficient technical solution for controlling this serious disease that currently threatens the goose farming industry.

[0062] This invention provides an egg yolk antibody for goose astrovirus extracted and purified from hyperimmune egg yolks. This egg yolk antibody has a high titer, a cure rate of over 80% for early-infected geese, a passive protection period of up to 8 days, and a good effect in preventing goose astrovirus infection, with an animal protection rate of 100%.

[0063] The method for propagating goose astrovirus provided by this invention first involves culturing the virus in retinal epithelial cells that are highly sensitive to goose astrovirus (increasing the viral titer in goose retinal epithelial cells by more than 100 times, thus reducing vaccine production costs). Then, the method of controlling variables is used to screen the excipient sodium butyrate to improve the replication efficiency of N-GoAstV-hs2403 in retinal epithelial cells, thereby providing high-titer viral fluid for vaccine production. Attached Figure Description

[0064] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 The image shows the RT-PCR identification of N-GoAstV-hs2403. In the image, M represents DNA marker, 1 represents goose astrovirus (GoAstV), 2 represents goose parvovirus (GPV), 3 represents reovirus (NDRV), 4 represents adenovirus (FAdV), 5 represents avian influenza virus (AIV), 6 represents Newcastle disease virus (NDV), 7 represents goose circovirus (GoCV), and 8 represents goose astrovirus (N-GoAstV).

[0065] Figure 2 IFA identification diagram of N-GoAstV-hs2403.

[0066] Figure 3 The results are from a response surface methodology experiment.

[0067] Figure 4 This is a sterility test image of inactivated goose astrovirus N-GoAstV-hs2403.

[0068] Figure 5 This is a safety verification diagram for the inactivated goose asteroid virus N-GoAstV-hs2403.

[0069] Figure 6 This is a diagram illustrating the immunization effect of the goose astrovirus inactivated vaccine. Detailed Implementation

[0070] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0071] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0072] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0073] Example 1: Isolation and identification of goose astrovirus N-GoAstV-hs2403 strain 1.1 Separation Liver tissue was collected from goslings with typical gout disease at a goose farm in Shandong Province. Five volumes of PBS were added, and the tissue blocks were ground and then added to PBS containing antibiotics to prepare a 10% suspension. The suspension was subjected to three freeze-thaw cycles at -80℃, centrifuged at 3000 rpm for 20 min, and the supernatant was filtered through a 0.22 μm filter. The supernatant was then inoculated into the allantoic cavity of 8-day-old SPF goose embryos at a rate of 0.2 mL / embryo and incubated at 37℃ for 72 h. Allantoic fluid from embryos that died within 72 h and from surviving embryos was harvested and stored at -80℃.

[0074] 1.2 Identification 1.2.1 RT-PCR identification The harvested viral fluid was subjected to RT-PCR to detect goose astrovirus, with goose parvovirus (GPV), goose reovirus (GRV), adenovirus (FAdV), avian influenza virus (AIV), Newcastle disease virus (NDV), and goose circovirus (GoCV) serving as control groups. The primers used were ORF2-F:5'-ATGGCTAGCTGCAGCGAC-3' (SEQ ID NO:1) and ORF2-R:5'-TCAGTGGCGGCCGCTCTAGA-3' (SEQ ID NO:2).

[0075] Upon identification, the isolated strain (designated N-GoAstV-hs2403) only amplified the corresponding target fragment of the astrovirus, which was consistent with the expected size of the target fragment. Figure 1 No other viruses were detected.

[0076] 1.2.2 IFA Qualification Inoculation: Goose retinal epithelial cells (Tibetan ID: CCTCCNO: C2021238) were cultured in 96-well plates for 24 hours and then inoculated with N-GoAstV-hs2403 virus at a volume of 0.1 mL per well.

[0077] Fixation: After inoculation, cells were cultured at 37°C and 5% CO2 for 72 h, followed by two washes with PBS. 100 μL of anhydrous ethanol was added to each well, and the cells were fixed at 4°C for 45 min. The anhydrous ethanol was discarded, and the cells were washed twice with 100 μL of PBS per well.

[0078] Primary antibody incubation: Dilute rabbit GoAstv polyclonal antibody (Nanjing Zhongding Biotechnology Co., Ltd., ZD2024H02372) 1:1000 with PBS, add 50 μL to each well, incubate at 37℃ for 1 h, and wash three times with PBS.

[0079] Secondary antibody incubation: In a dark environment, dilute FITC-labeled goat anti-rabbit IgG (H+L) fluorescent antibody (Shanghai Yisheng Biotechnology Co., Ltd., BD1044184) 1:500 with PBS, adding 40 μL of the diluted antibody to each well. Wrap in aluminum foil and incubate at 37°C in the dark for 1 hour, then wash three times with PBS. Observe under an inverted fluorescence microscope using blue excitation light.

[0080] IFA assessment results are as follows: Figure 2 As shown.

[0081] 1.2.3 Animal reintroduction experiment N-GoAstV-hs2403 virus solution (FAID) 50 =10 8.50 0.5 mL / gosling was administered orally to healthy 3-day-old goslings. Observation of morbidity and mortality was conducted, and necropsy was performed on each gosling.

[0082] The results showed that the morbidity rate of goslings reached 100% and the mortality rate was 20% within 5 days. Autopsy revealed typical urate deposits in the internal organs. The same virus was re-isolated from the dead geese, thus verifying Koch's postulates.

[0083] Through the above experiments, this invention obtained a highly pathogenic goose astrovirus N-GoAstV-hs2403, with accession number CCTCC NO:V202561, accession date August 22, 2025, and accession address China Center for Type Culture Collection.

[0084] Example 2: Optimization of passage culture conditions for goose astrovirus N-GoAstV-hs2403 1. First round of screening for virus propagation process Goose retinal epithelial cells (CCTCC NO: C2021238, hereinafter the same) were cultured for 72 h, then the cell culture medium was discarded, and N-GoAstV-hs2403 virus was inoculated. The experimental procedure was designed using a controlled single-factor variable method, and the details are as follows: Each T25 flask was inoculated with virus at MOIs of 0, 0.005, 0.01, 0.02, and 0.04. In all five groups, 50 μg of TPCK trypsin (gibco, LOT 2277575) was added. A maintenance medium (serum-free DMEM-F12 medium, Vigonob, Cat NO 66118-18516) was added to a volume of 5 mL. The cells were incubated at 37°C with 5% CO2 for 2 h for adsorption. Then, a total maintenance medium (serum-free DMEM-F12 medium) of 10 mL was added. The virus was cultured for 80-90 h and harvested. The harvested virus solution was repeatedly frozen and thawed three times before virus titer detection. The virus was serially diluted 10-fold after three freeze-thaw cycles, with the dilution range being 10. -1 -10 -8 The virus was inoculated at a dilution of 100 μL into 96-well plates lined with goose retinal epithelial cells. After 4 days, the FAID was calculated using the Reed-Mucnch method by indirect fluorescence (IFA detection). 50 The results are shown in Table 1. When the MOI of the inoculation amount was 0.01, the highest virus titer harvested after 80-90 hours of virus culture was 10. 7.20 FAID 50 / mL (see Table 1 for results) is more effective and is the best virus propagation process for screening.

[0085] Table 1. Results of virus propagation process screening

[0086] 2. Screening of virus propagation processes After culturing goose retinal epithelial cells for 72 hours, the cell culture medium was discarded, and N-GoAstV-hs2403 inoculation was performed. The design followed a controlled single-factor variable method, detailed below: Five experimental groups were formed. In each group, T25 flasks were inoculated with an MOI of 0.01, TPCK trypsin was added at 50 μg, and maintenance medium (serum-free DMEM-F12 medium, Vigonob, Cat NO 66118-18516) was added to a volume of 5 mL. The cells were incubated at 37°C with 5% CO2 for 2 hours. Afterward, maintenance medium (serum-free DMEM-F12 medium) was added to a total volume of 10 mL. Virus culture was continued for 12-18 hours, and sodium butyrate was added to the five experimental groups at concentrations of 0 mmol / L, 1 mmol / L, 2.5 mmol / L, 5 mmol / L, and 10 mmol / L, respectively. The virus was then cultured for 80-90 hours before harvesting. The harvested virus solution was subjected to three freeze-thaw cycles before virus titer detection. The virus was serially diluted 10-fold after three freeze-thaw cycles, with the dilution range being 10. -1 -10 -8 The virus was inoculated at a dilution of 100 μL into 96-well plates lined with goose retinal epithelial cells. After 4 days, the FAID was calculated using the Reed-Mucnch method by indirect fluorescence (IFA detection). 50 .

[0087] The results are shown in Table 2. The results indicate that the highest harvest titer of 10 was achieved when the MOI (Mean Infection Index) was 0.01, the TPCK trypsin dosage was 50 μg, the sodium butyrate dosage was 2.5 mmol / L, and the virus was cultured for 80-90 h. 8.20 FAID 50 / mL, as the optimal virus propagation process for screening.

[0088] Table 2 Results of virus propagation process screening

[0089] 3. Screening of virus propagation processes After culturing goose retinal epithelial cells for 72 hours, the cell culture medium was discarded, and N-GoAstV-hs2403 inoculation was performed. The design followed a controlled single-factor variable method, detailed below: Five experimental groups were formed. In each group, T25 flasks were inoculated with an MOI of 0.01. TPCK trypsin was added at 50 μg. Maintenance medium (serum-free DMEM-F12 medium, Vigonob, Cat NO 66118-18516) was added to a volume of 5 mL. Cell culture was incubated at 37℃ with 5% CO2 for 0, 0.5, 1, 2, and 4 hours, followed by replenishment of maintenance medium (serum-free DMEM-F12 medium) to a total volume of 10 mL. When the virus cultured for 12-18 hours, sodium butyrate at 2.5 mmol / L was added. The virus was then cultured for 80-90 hours before harvesting. The harvested virus solution was subjected to three freeze-thaw cycles before virus titer detection. The virus was serially diluted 10-fold after three freeze-thaw cycles, with the dilution range being 10. -1 -10 -8 The virus was inoculated at a dilution of 100 μL into 96-well plates lined with goose retinal epithelial cells. After 4 days, the FAID was calculated using the Reed-Mucnch method by indirect fluorescence (IFA detection). 50 The results showed that the highest harvest titer of 10 was achieved when the MOI of virus inoculation was 0.01, the amount of TPCK trypsin added was 50 μg, the amount of sodium butyrate added was 2.5 mmol / L, the virus adsorption time was 2 h, and the virus culture time was 80-90 h. 8.50 FAID 50 / mL (see Table 3 for results) was used as the optimal virus propagation process for screening.

[0090] Table 3. Results of virus propagation process screening

[0091] 4. Response Surface Design To determine the optimal experimental conditions and interactions among factors for inoculation with N-GoAstV-hs2403 strain, a Box-Behnken experimental design was employed, with FAID as the primary factor. 50 The highest value was taken as the response value. A total of 9 factorial experiments were conducted with 3 factors and 3 levels to further optimize and screen for the optimal key parameters. The factors and levels are shown in Table 4.

[0092] Table 4 Factors and levels in Box-Behnken experimental design

[0093] Response surface methodology results are as follows Figure 3As shown, the response surface plot is cone-shaped, indicating the existence of extreme values. Based on the changes in the response surface, the optimal parameters for MOI, sodium butyrate addition, and adsorption time are 0.011, 2.458 mmol, and 2.2 h, respectively. Under these conditions, the FAID is predicted. 50 / 0.1mL=8.67293(R 2 =0.995), which is twice as high as the basic culture.

[0094] 5. Confirmation of optimal conditions for virus reception Repeat the experimental conditions confirmed in step 4 three times to confirm the optimal conditions for virus inoculation.

[0095] The optimal virus propagation process is as follows: After culturing goose retinal epithelial cells for 72 hours, the cell culture medium was discarded, and N-GoAstV-hs2403 virus was inoculated. The inoculation volume in T25 square bottles was set at an MOI of 0.01, and 50 μg of TPCK trypsin was added. The volume of maintenance medium, i.e., serum-free DMEM-F12 medium (Vigonob, Cat NO 66118-18516), was added to 5 mL. After adsorption time in a cell culture incubator at 37°C and 5% CO2 for 2 hours, the volume of maintenance medium, i.e., serum-free DMEM-F12 medium, was added to 10 mL. The virus was cultured for 12-18 hours, and sodium butyrate at 2.5 mmol / L was added. The virus was cultured for 80-90 hours, and the virus solution was harvested.

[0096] Example 3: Preparation of inactivated goose astrovirus vaccine A method for preparing an inactivated goose astrovirus vaccine includes the following steps: (1) Viral proliferation Take the 18th generation goose astrovirus N-GoAstV-hs2403 strain and dilute it to ELD. 50 =5×10 5.0 / mL, inoculated into the allantoic cavity of 9-day-old SPF goose embryos, 0.2mL / embryo, and incubated at 37℃ for 72h. Embryos that die within 24h are discarded; allantoic fluid from surviving embryos is harvested, mixed, centrifuged at 3000r / min for 20min, and filtered through a 0.45μm filter membrane. The toxicity titer is determined to be ELD. 50 =10 6.3 0 / mL.

[0097] (2) Virus inactivation In step (1), 10% formaldehyde solution was added to the filtered virus solution to bring the final concentration to 0.2%, and the virus was inactivated by magnetic stirring at 37°C for 24 hours. Samples were then taken for testing. Sterility test: The inactivated virus solution was inoculated into TSB, GA, and FTM media and incubated at 37°C for 7 days. Results are as follows: Figure 4As shown, no microorganisms grew on TSB, GA, and FTM media.

[0098] Inactivation validation: Twenty healthy goose embryos aged 9-11 days were randomly divided into two groups of 10 each. The first group (experimental group) was injected with the prepared inactivated virus solution, 0.2 mL / embryo. The second group (control group) was injected with an equal volume of PBS. After incubation at 37°C for 72 hours, embryo survival was observed. If the embryos were healthy, they were blindly passaged for one generation. If the embryos remained healthy, complete inactivation was confirmed, indicating good safety of the prepared inactivated virus solution. Results are as follows: Figure 5 As shown, the prepared inactivated vaccine has good safety.

[0099] (3) Vaccine emulsification Oil phase preparation: Mix white mineral oil and Span at a mass ratio of 94.5%:5.5% thoroughly and sterilize at 121°C for 30 minutes for later use.

[0100] Aqueous phase preparation: Inactivated antigen (i.e. virus solution) is added to the tank at a mass ratio of 97.5%, and 2.5% sterilized Tween is added at the same time. Stir thoroughly until the Tween is completely dissolved.

[0101] Emulsification: The aqueous phase and oil phase are mixed at a volume ratio of 1:2. First, the oil phase is injected into the emulsification tank and stirred. Then, the aqueous phase is slowly added and stirred to ensure that the oil and water phases are fully mixed. Finally, the mixture is emulsified online through a shearing machine to obtain the goose astrovirus inactivated vaccine.

[0102] The inactivated goose astrovirus vaccine prepared above was stored at 4℃ and 25℃ respectively. Samples were taken monthly to test the agar amplification titer of the inactivated goose astrovirus vaccine. The agar amplification detection method was referred to (Wei Xinyu. Establishment and preliminary application of anti-goose astrovirus antibody detection method [D]. Nanjing Agricultural University, 2022. DOI:10.27244 / d.cnki.gnjnu.2022.001227.), viscosity, and bacterial contamination.

[0103] The results showed that the agar amplification titer of the goose astrovirus inactivated vaccine was 1:256. It was sterile and grew within 12 months of storage at 4℃, with a decrease in agar amplification titer of <5% and no significant change in viscosity. It was sterile and grew within 6 months of storage at 25℃, with a decrease in titer of about 15% and a slight increase in viscosity, but it could still be injected smoothly. The agar amplification titers are shown in Table 5.

[0104] Table 5. Amplified potency of goose astrovirus inactivated vaccine (Vaccine)

[0105] Example 4: Evaluation of the efficacy of the goose astrovirus inactivated vaccine 1. Serological methods The inactivated goose astrovirus vaccine prepared in Example 3 was used to immunize 30-day-old goslings (n=20) at a dose of 1.0 mL / bird, with a 14-day interval between the second immunization. A control group (n=20) was also included. Blood samples were collected on days 7, 14, 21, 28, 35, 42, and 79 after immunization. Serum was separated, and the neutralizing antibody titer of goose astrovirus in the serum was determined. The detection method was based on (Song Rui. Isolation, Identification, and Antibody Detection Methods for Chicken Astrovirus in Huai'an Area, Jiangsu Province [D]. Yangzhou University, 2023. DOI:10.27441 / d.cnki.gyzdu.2023.002370.).

[0106] Test results as follows Figure 6 As shown, the inactivated goose astrovirus vaccine prepared in Example 3 can produce a high titer of antibody levels and has a certain preventive effect against the virus.

[0107] 2. Immune challenge method Immunization: 2-day-old Magang geese were subcutaneously inoculated with 0.3 mL of GoAstV inactivated vaccine in their necks. After immunization, the geese's diet, water intake, and local and systemic reactions were observed daily. Serum samples were collected from each group of geese at 14, 21, and 28 days after immunization to measure the GoAstV agar amplification titer.

[0108] Challenge: 28 days post-immunization, each goose in both the immunized and challenged groups was challenged with 2.0 mL of undiluted GoAstV-2301 virus or a 10-fold dilution via eye drops or oral administration. The geese were observed for 17 days post-challenge, primarily for signs such as lethargy, decreased appetite, increased water intake, weight loss, limb paralysis, and white, watery stools. Throat and cloacal swabs were collected from both groups on days 3, 5, 7, 10, 12, 14, and 17 post-challenge for PCR testing to detect virus shedding. Simultaneously, 5 days post-challenge, one goose from each group was randomly selected for necropsy to observe visceral lesions, primarily for signs of visceral swelling and urate deposition. Seventeen days after the challenge, serum was collected from each group of experimental geese to monitor the neutralizing titer of GoAstV. All experimental geese were also necropsyd to observe for mottled kidneys and urate deposits in the ureters, joint cavities, heart, liver, and other parts of the body. The results were also photographed and recorded.

[0109] 3. Vaccine safety trials Twenty healthy 7-day-old goslings were randomly divided into two groups of 10 each. The first group was the experimental group, which received a subcutaneous injection of 1 mL of the inactivated goose astrovirus vaccine prepared in Example 3 into the neck. The second group was the control group, which received an equal volume of PBS subcutaneously into the neck. The goslings were observed for 14 consecutive days after vaccination.

[0110] The results showed that the weight gain of goslings in the experimental group and the control group was similar, the goslings were in good spirits and had normal diets, and no adverse reactions such as redness, swelling and necrosis occurred at the injection site, indicating that the goose astrovirus inactivated vaccine prepared in Example 3 had good safety.

[0111] Example 5 Preparation of Egg Yolk Antibodies A method for preparing egg yolk antibodies includes the following steps: (1) Immunization of laying hens Fifty 120-day-old Hy-Line Brown laying hens were selected, and the immunization program was as follows: Primary immunization: 1.0 mL / bird of the inactivated goose astrovirus vaccine prepared in Example 3, subcutaneously injected into the neck; Second immunization: 14 days later, 1.5 mL / bird, subcutaneously injected into the neck; Third immunization: 14 days later, 1.5 mL / bird, subcutaneously injected into the neck; Maintenance immunization: 1.5 mL / bird booster when the yolk sac agar titer is ≥1:96.

[0112] (2) Collection of high immunity eggs After the fourth vaccination, antibody titers are tested every seven days. Eggs are collected once the antibody titer reaches 1:2000. Eggs are disinfected by soaking in 0.1% benzalkonium chloride for 20 minutes, wiped with 75% alcohol, and the yolks are separated in a clean bench to obtain yolk solution, which is then stored at -20℃ for later use.

[0113] (3) Antibody extraction 1) Acidification extraction: Take 100 mL of egg yolk fluid, add 800 mL of pH 5.2 acetate buffer (0.15 M NaCl, 0.02 M sodium acetate), stir at 4℃ for 2 h, let stand overnight, centrifuge at 10000 r / min for 10 min, and take the supernatant; 2) Ammonium sulfate precipitation First precipitation: Add an equal volume of saturated ammonium sulfate to the supernatant until 50% saturation, precipitate overnight at 4°C, and centrifuge at 8000 r / min for 20 min; Secondary precipitation: The precipitate was dissolved in 100 mL PBS, and 200 mL saturated ammonium sulfate was added to a saturation of 66.7%. The precipitate was precipitated at room temperature for 24 h. After centrifugation, the precipitate was dissolved in 50 mL PBS. 3) Dialysis: The solution was placed in a 10kD dialysis bag and dialyzed against 0.85% NaCl for 24 hours. The solution was changed 3 times, and then dialyzed against PBS for 12 hours.

[0114] (4) Refining and Preparation 1) Ultrafiltration sterilization: The dissolved solution after dialysis is passed sequentially through a 0.45μm filter membrane and a 1000kD ultrafiltration membrane; 2) Concentration: A 100kD hollow fiber column is concentrated 10 times to approximately 10mL; 3) Inactivation: Add 0.05% formaldehyde and stir at room temperature for 24 hours; 4) Freeze-drying: Egg yolk antibody and protectant are mixed in a 1:1 ratio in the freeze-dried powder mixture system. The protectant formula includes 40% trehalose, 30% sucrose, 20% mannitol, 5% glycine, and 5% arginine. After dispensing, the mixture is freeze-dried (see Table 6) to obtain egg yolk antibody freeze-dried powder.

[0115] Table 6. Lyophilization conditions for egg yolk antibodies (process parameters)

[0116] 5) Discharge and storage: After freeze drying, quickly cap the product or vacuum seal it in an aluminum-plastic composite bag in a dry and clean environment (relative humidity ≤ 30%), store it at 4 ℃ away from light, and the shelf life is ≥ 18 months. The egg yolk antibody activity retention rate can reach more than 90%, and the product is clear and free of precipitation after reconstitution.

[0117] Example 6: Evaluation of the efficacy of egg yolk antibodies 1. Determination of egg yolk antibody titer Melt 1% agar and pour it into 100×15 mm petri dishes (2.5–3 mm thick). After solidification at room temperature, punch 7 wells in a quincunx pattern and remove the agar plug. For the surrounding 6 wells: add 20 µL of egg yolk antibody at different dilutions clockwise, one dilution per well; also include one well each for positive, negative, and antigen controls. Observe the results within 24–48 hours at 37℃. A positive result is defined as the appearance of a clear white precipitate line that matches the positive control line. The highest dilution forming a visible precipitate line is the AGID titer of the egg yolk antibody.

[0118] The results showed that 7 days after the third immunization, the agar amplification titer of egg yolk antibody was 1:128; at the peak (30 days after the third immunization): the agar amplification titer was 1:1024; and at the maintenance period (150 days after the third immunization): the agar amplification titer was 1:128.

[0119] 2. Passive immunization phase assay for antibodies Healthy, susceptible goslings aged 1 day (n=60) were artificially infected with goose astrovirus N-GoAstV-hs2403 for 12 hours and then randomly divided into three groups: a treatment group, a challenge control group, and a blank control group. The treatment group received an intramuscular injection of egg yolk antibody solution (the lyophilized egg yolk antibody powder was diluted to 1 mg / mL with 0.01 M pH 7.2-7.4 PBS solution) filtered through a 0.22 µm sterile filter, with an injection dose of 1.0 mL per gosling. The control group received an intramuscular injection of 0.01 M pH 7.2-7.4 PBS solution. The recovery of goslings in each treatment group was observed.

[0120] The results showed that the recovery rate in the treatment group reached 80% (16 / 20) in 8 days, with an average recovery time of 3.2 days; the mortality rate in the challenge control group reached 90% (18 / 20) in 8 days. The passive antibody immunization data are shown in Table 7.

[0121] Table 7. Antibody Passive Immunization Data

[0122] 3. Safety test of egg yolk antibodies Forty healthy, one-day-old susceptible goslings were randomly divided into two groups. The experimental group received an intramuscular injection of 1.0 mL of egg yolk antibody per gosling, while the control group received an intramuscular injection of the same volume of physiological saline. For 14 consecutive days, their mental status, feed intake, body weight, number of deaths, local redness and induration, and necropsy findings were recorded. All goslings survived without any local or systemic abnormalities.

[0123] In summary, this invention successfully isolated and identified the N-GoAstV-hs2403 strain of goose astrovirus, and prepared a high-titer goose astrovirus solution using this strain, with a titer of FAID. 50 =10 8.50 / mL, the yolk antibody titer prepared afterward is high, and it has the best effect in preventing goose astrovirus infection, with an animal protection rate of 100%.

[0124] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A goose astrovirus strain, named N-GoAstV-hs2403, was deposited at the China Center for Type Culture Collection on August 22, 2025, with accession number CCTCC NO:V202561.

2. The use of the goose astrovirus according to claim 1 in the preparation of products for the prevention and control of diseases caused by goose astrovirus.

3. The application according to claim 2, characterized in that, The products include reagents, drugs, and / or egg yolk antibodies; Preferably, the reagent includes diagnostic reagents; Preferably, the drug includes a vaccine; Preferably, the vaccine comprises an inactivated vaccine.

4. A vaccine, characterized in that, The active ingredient of the vaccine includes the inactivated virus of the goose astrovirus as described in claim 1.

5. The vaccine according to claim 4, characterized in that, The vaccine also includes pharmaceutically acceptable excipients; Preferably, the excipients include at least one of a carrier, a protective agent, an adjuvant, a formaldehyde, or a diluent; Preferably, the excipients include Tween, white oil, and Span.

6. The method for preparing the vaccine according to claim 5, comprising the following steps: The goose astrovirus of claim 1 is cultured and proliferated in goose retinal epithelial cells to obtain viral fluid; or the goose astrovirus is inoculated into SPF goose embryos, incubated, and the allantoic fluid of the surviving embryos is collected to obtain viral fluid. Inactivation yields an inactivated virus solution; The inactivated virus solution was mixed with Tween to obtain an aqueous phase; The white oil and Span were mixed to obtain the oil phase; The oil and water phases are mixed and emulsified to obtain the vaccine.

7. The preparation method according to claim 6, characterized in that, The culture and proliferation process includes the following steps: goose astrovirus is inoculated into goose retinal epithelial cells at an MOI of 0.005-0.04; TPCK trypsin is added to the culture system and adsorbed in a cell culture incubator; cell maintenance medium is added and cultured for 10-20 hours; sodium butyrate is added to the culture system and cultured for 70-90 hours to obtain the virus solution.

8. A goose astrovirus yolk antibody obtained by immunizing laying hens with the vaccine described in claim 4 or 5; Preferably, the goose astrovirus yolk antibody is prepared by a method comprising the following steps: immunizing laying hens with the vaccine; collecting hyperimmune eggs and separating the yolks; and extracting the yolks by acidification and precipitation with ammonium sulfate to obtain the goose astrovirus yolk antibody.

9. A method for propagating goose astrovirus, comprising inoculating goose retinal epithelial cells with the goose astrovirus of claim 1, adding TPCK trypsin to the culture system and adsorbing it in a cell culture incubator; supplementing with cell maintenance medium and culturing for the first time; adding sodium butyrate to the culture system and culturing for the second time.

10. The method according to claim 9, characterized in that, The final concentration of TPCK trypsin in the culture system is 5-15 μg / mL; Preferably, the goose astrovirus is inoculated at an MOI of 0.005-0.04; Preferably, the final concentration of sodium butyrate in the culture system is 1-10 mmol / L.

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