Goose astrovirus and application thereof in preparation of vaccine

Abstract

The invention aims to provide a goose astrovirus and an application of the goose astrovirus in preparation of vaccines, the goose astrovirus is named as goose astrovirus GAstV-M705, the taxonomic name of the goose astrovirus is Avastrovirus-Goose astrovirus, the goose astrovirus is preserved in the China Center for Type Culture Collection, the preservation number is CCTCC NO: V202521, and the preservation date is April 29, 2025. The strain is subjected to passage and proliferation on goose embryos and LMH cells, it is found that the strain can be stably proliferated, then the strain serves as an antigen to be prepared into the inactivated vaccine, and a challenge protection test proves that the inactivated vaccine has good immunogenicity.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a goose astrovirus and its application in preparing vaccines. Background Art

[0002] As a key species in my country's agricultural waterfowl industry, goose farming holds irreplaceable strategic value. In recent years, outbreaks of a disease characterized by urate accumulation in various organs and joints have occurred in goose farms in parts of my country, causing significant losses to the goose industry. A comprehensive approach, including virus isolation and culture, and validation in animal infection models, ultimately identified the causative agent of this disease as goose astrovirus (GAstV). The disease is highly contagious. Once initiated, it often spreads throughout the entire goose farm. Infected breeders can transmit the disease vertically to embryos, resulting in flock mortality exceeding 50% in goslings within four weeks of age, causing significant economic losses to the Chinese goose industry. Although numerous studies have reported on vaccines for GAstV, there are currently no commercially available vaccines or effective treatments for GAstV. Therefore, continued and in-depth research on GAstV is essential. Summary of the Invention

[0003] In response to the current difficulties in the development of goose astrovirus vaccines, the present invention intends to isolate and identify goose astrovirus. Given the characteristics of inactivated vaccine preparations, such as controllable biosafety, excellent antigenic epitope integrity, and strong adaptability to large-scale production, the present invention further selects isolated strains to construct an inactivated antigen system. By analyzing its immunogenicity, the system provides a practical basis for disease prevention and control and vaccine development. The technical solutions of the present invention are as follows: In the first aspect, the present invention provides a goose astrovirus, which is named goose astrovirus GAstV-M705 and its taxonomic name is Avastrovirus-Goose astrovirus , deposited in the China Center for Type Culture Collection, the deposit number is CCTCC NO: V202521, and the deposit date is April 29, 2025.

[0004] In a second aspect, the present invention provides use of the goose astrovirus described in the first aspect in preparing a vaccine for preventing and treating goose astrovirus.

[0005] In a third aspect, the present invention provides an inactivated vaccine for preventing and treating goose astrovirus, wherein the inactivated vaccine contains the goose astrovirus described in the first aspect.

[0006] Preferably, the goose astrovirus adopts cell virus liquid of passage 5 or above.

[0007] More preferably, the goose astrovirus adopts cell virus liquid of the sixth passage or above.

[0008] More preferably, the TCID50 of the cell virus solution is greater than 10-6 / 0.1 mL.

[0009] In a fourth aspect, the present invention provides a method for preparing the inactivated vaccine according to the third aspect, comprising the following steps: Step 1, inactivating the goose astrovirus liquid to obtain an antigen liquid; Step 2, preparing an oil phase and an aqueous phase respectively, wherein the preparation of the oil phase comprises: premixing a medicinal white oil with an emulsifier, adding a stabilizer, heating the mixture to a temperature of 80-120° C. in a gradient manner, mixing the mixture until a homogeneous transparent phase is formed, and then sterilizing the mixture to obtain the oil phase; The preparation of the aqueous phase comprises: mixing the antigen solution with sterile Tween-80, separating the insoluble matter, and obtaining the aqueous phase; Step 3: mixing the oil phase and the water phase in a volume ratio of 2 to 3:1 to obtain the inactivated vaccine.

[0010] Preferably, in step 1, the inactivation method includes: using formaldehyde as an inactivating agent, and the inactivation time is 20 to 40 hours; preferably, the amount of formaldehyde added is 0.3% of the volume of the virus liquid, and the inactivation time is 20 to 24 hours.

[0011] Preferably, in step 2, the medicinal white oil and the emulsifier are premixed in a volume ratio of 93-97:3-7, and the amount of the stabilizer added is 1-4% of the total mass of the oil phase; and / or, the antigen solution and sterile Tween-80 are mixed in a ratio of 30-40:1-2; and / or, the separation of insoluble matter is performed by centrifugation. Preferably, the control parameters of the centrifugation include: 4000-8000 rpm, and a centrifugation time of 10-20 min.

[0012] Preferably, in step 3, the method of mixing the oil phase and the water phase comprises: adding the water phase to the oil phase at a mixing speed of 400-600 rpm, adjusting the mixing speed to 800-1200 rpm after the addition, continuing the homogenization for 6-10 minutes, with an interval of 3 minutes, and repeating 3 times continuously to fully blend the two phases.

[0013] Compared with the prior art, the technical effects of the present invention are: The present invention clinically isolated a GAstV M705 strain, and further passaged and propagated the strain on goose embryos and LMH cells, and found that the strain can stably proliferate. The phylogenetic evolutionary tree and homology show that the M705 strain belongs to the same genotype II as most GAstV strains, and its homology with the typical type I strains FLX and AHDY is only 57.5%. The present invention further uses it as an antigen to prepare an inactivated vaccine. The challenge protection test confirms that the inactivated vaccine has stable properties, good safety, and good immunogenicity, and is expected to provide an important reference for the development of GAstV-related vaccine products in the domestic market. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings: Figure 1 These are the PCR / RT-PCR test results of the diseased materials in Example 1 of the present invention. In Figure A, M is DL 2000 Marker, and lanes 1-7 correspond to the test results of goose circovirus, avian adenovirus, Newcastle disease, goose parvovirus, Tembusu virus, avian influenza virus, and avian reovirus, respectively; in Figure B, M' is DL 2000 Marker, abc are the test results of goose astrovirus in the liver, spleen, and kidney of the tested goose, respectively, and d is a negative control.

[0015] Figure 2 The RT-PCR results of goose embryo allantoic fluid in Example 1 of the present invention are shown, wherein M is DL2000 DNA Marker; 1-6 are allantoic fluids from the sixth to first generations; 8 is a positive control; and 7 is a negative control.

[0016] Figure 3 The growth curve and virus titer change curve of the GAstVM705 strain on LMH cells in Example 1 of the present invention are shown, wherein A is the growth curve, showing the change in virus titer over time; B is the change in virus titer during continuous passage of the strain on LMH cells.

[0017] Figure 4 The electron micrographs of goose astrovirus in Example 1 of the present invention are shown, wherein A is the virus particles observed under a low-power microscope (15,000 times); and B is the virus particles observed under a high-power microscope (80,000 times).

[0018] Figure 5 The toxicity of the virus in Example 1 of the present invention changes when it is treated with some physical and chemical factors.

[0019] Figure 6The figure shows the trend of body weight changes of goslings after challenge with poison in Example 1 of the present invention. The results are expressed as mean ± standard deviation (SD), n = 20. Asterisks indicate significant differences among the groups (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

[0020] Figure 7 The PCR results of the whole genome segmented amplification of the GAstV M705 strain in Example 3, wherein M is the DL 2000 Marker, and 1-6 are the amplification products of the first to sixth pairs of primers, respectively.

[0021] Figure 8 The phylogenetic tree of the GAstV whole genome and each ORF in Example 3, wherein A is the whole genome evolutionary tree, B is the ORF1a evolutionary tree, C is the ORF1b evolutionary tree, and D is the ORF2 evolutionary tree.

[0022] Figure 9 This is a comparison diagram of the whole genome nucleotide homology in Example 3.

[0023] Figure 10 This is a comparison diagram of the nucleotide homology of ORF1a in Example 3.

[0024] Figure 11 This is a comparison diagram of the nucleotide homology of ORF1b in Example 3.

[0025] Figure 12 This is a comparison diagram of the ORF2 nucleotide homology in Example 3.

[0026] Figure 13 For the detection of GAstV-specific antibody levels in serum in Example 4, one-way ANOVA was used to analyze the significance of the differences. Very significant differences were indicated by "****" (P < 0.0001); extremely significant differences were indicated by "***" (P < 0.001); extremely significant differences were indicated by "**" (P < 0.01); significant differences were indicated by "*" (P < 0.05); and not significant differences were indicated by "ns" (P > 0.05).

[0027] Figure 14 For the neutralizing antibodies in the sera of each group in Example 4, one-way ANOVA was used to analyze the significance of the differences. The differences were very significant and were indicated by "****" (P < 0.0001); the differences were extremely significant and were indicated by "***" (P < 0.001); the differences were extremely significant and were indicated by "**" (P < 0.01); the differences were significant and were indicated by "*" (P < 0.05); and the differences were not significant and were indicated by "ns" (P > 0.05). DETAILED DESCRIPTION

[0028] This study aims to isolate and identify goose astroviruses, studying their biological properties, including physicochemical characteristics and the complete viral genome sequence, to clarify their molecular epidemiological characteristics, genomic structure, and taxonomic status. Given the controllable biosafety, excellent antigenic epitope integrity, and strong suitability for large-scale production of inactivated vaccine formulations, an isolated strain was selected to construct an inactivated antigen system. By analyzing its immunogenicity, this system will lay a theoretical foundation for disease prevention and control and vaccine development.

[0029] In order to explore the cause of the disease, the present invention first extracted RNA from the diseased goslings and performed RT-PCR identification. The results preliminarily showed that it was GAstV. After grinding, it was inoculated into goose embryos and chicken liver cancer cells (LMH) for isolation, and the growth characteristics of GAstV on goose embryos and LMH cells were explored, and its related physical and chemical properties were explored. The virus was purified and the morphology of its virus particles was observed under an electron microscope. Animal regression experiments were performed to observe whether the symptoms of gosling gout could be artificially reproduced. The isolated strain was inoculated into 9-day-old goose embryos. As the number of passages increased, it could gradually and stably kill the goose embryos. The symptoms were redness and swelling of the embryo body, obvious bleeding spots in the organs, and obvious thickening of the chorioallantoic membrane. Specific bands appeared in the allantoic fluid. When the positive allantoic fluid was inoculated into LMH cells, cytopathic effect (CPE) could be produced. The cells became round and fell off, and the light-shielding ability decreased. As the number of passages increased, the virus titer gradually increased. By about the 10th generation, the TCID50 was stable at 10 -6 / 0.1ml or more; under transmission electron microscopy, the virus particles are approximately spherical, without envelope, and about 30nm in diameter, which is consistent with the typical shape of astrovirus.

[0030] The present invention uses isolated strains as antigens to explore the inactivation effects of different concentrations of formaldehyde inactivation at different times. The results show that 0.3% formaldehyde inactivation for 24h can completely inactivate the virus, so this condition is selected for inactivation. After the antigen and adjuvant are fully emulsified, they are prepared into inactivated vaccines. The appearance, dosage form, stability, viscosity, sterility and safety of the vaccine are tested. The results show that the dosage form is oil-in-water type, the physical properties are stable, the sterility test is qualified, the safety is good, the immune goslings do not die, the injection site is well absorbed and no inflammation occurs. The prepared vaccine is immunized to goslings, and the GAstV-specific antibody level in the goslings is detected by ELISA. The results show that the antibody level of each immune group begins to rise at 7 days and reaches a peak at 21 days. The antibody level is approximately positively correlated with the immune dose and then slightly decreases. The trace virus neutralization experiment detects the neutralizing antibody level. The average titer of the neutralizing antibody in the immune group can reach 9.01log2 at most, and no neutralizing antibody is produced in the PBS control group. In the attack and protection test, deaths occurred in the control group and the detoxification time was longer, while only the 0.2ml group in the immune group had deaths, and only sporadic positive samples were detected in other groups, indicating that 0.5ml can play a better protective role.

[0031] Furthermore, a goose astrovirus obtained in the present invention is named goose astrovirus GAstV-M705, and its taxonomic name is Avastrovirus-Goose astrovirus , deposited in the China Center for Type Culture Collection, the deposit number is CCTCC NO: V202521, the deposit date is April 29, 2025, and the address of the deposit center is: Wuhan University, Wuhan, China.

[0032] Furthermore, in the description of the present invention, it should be noted that, if specific conditions are not specified in the examples, the experiments were carried out according to conventional conditions or the conditions recommended by the manufacturer. Reagents or instruments used without specifying the manufacturer are all conventional products that can be purchased commercially.

[0033] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0034] Example 1:

[0035] The isolation and identification of goose astrovirus are as follows: 1.1 Treatment and storage of diseased materials Source of experimental animals: In 2023, 15-day-old Sichuan white goslings at a goose farm in Sichuan showed symptoms such as decreased appetite, unstable gait, paralysis, depression, and white loose stools, and some died. Autopsy and examination of the sick geese revealed enlarged organs and white urate deposits.

[0036] The processing and preservation method is as follows: aseptically collect obviously diseased tissues such as kidney, liver, and spleen, cut a part and weigh about 0.5g, put it into a sterile centrifuge tube, add 1.5ml PBS, mince until no obvious tissue clumps can be seen with the naked eye, use a tissue homogenizer to mix and grind the diseased material, take the supernatant, and then transfer it to a new 2mL centrifuge tube, place the sample to be processed in a -80℃ refrigerator for 12 hours, transfer it to room temperature and completely thaw, repeat this freeze-thaw cycle 3 times to release the viral nucleic acid in the cells, add 100μL of double antibody, centrifuge at 4℃ and 8000r / min for 20min, take the supernatant and pass it through a 0.22μm sterilization filter, divide it into another centrifuge tube, and store the obtained virus liquid in a -80℃ refrigerator.

[0037] 1.2 Identification of diseased materials 1.2.1 Viral RNA extraction The RNA virus genome extraction kit of FOREGENE was used and the operation was performed according to its instructions, which will not be described in detail here.

[0038] 1.2.2 cDNA synthesis Using FOREGENE's 2×RT OR-Easy TM cDNA was synthesized by mixing the mixture and the RNA extracted in step 1.2.1 in a 1:1 ratio. The mixture was placed in a PCR instrument and the program was set to 42°C for 15 min, 85°C for 5 min, and long-term storage at 4°C. After the reaction, the cDNA was stored at -20°C until use. If long-term use is required, it should be stored at -80°C. The reaction system is shown in Table 1.

[0039] Table 1 Reverse transcription reaction system

[0040] 1.2.3 Extraction of viral DNA The DNA viral genome extraction kit of Solebio Biotech was used and the operation was performed according to the instructions. A detailed description is not given here.

[0041] 1.2.4 Virus Detection (1) Primer design In view of the viral diseases that often occur in goose flocks in my country, these common viruses were detected, including avian influenza virus (AIV), reovirus (ARV), Tembusu virus (ATMUV), goose parvovirus (GPV), goose circovirus (GoCV), avian adenovirus (FAdV), Newcastle disease virus (NDV) and goose astrovirus (GAstV). The primer sequences are shown in Table 2.

[0042] Table 2 Identification primers for different viruses

[0043] (2) Amplification of viral gene fragments Use the cDNA obtained in 1.2.2 and the DNA obtained in 1.2.3 as templates and perform PCR amplification using the specific primers in Table 2. The reaction system is 12.5 μl and the reaction system is as shown in Table 3. The reaction conditions are as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 15 s, for 35 cycles; extension at 72°C for another 10 min; storage at 4°C, until ∞.

[0044] Table 3 Reaction system

[0045] (3) Agarose gel electrophoresis Weigh 1.0 g of agarose powder into an Erlenmeyer flask and add 100 mL of pre-prepared 1× Tris-acetate-EDTA buffer (TAE working solution). Heat in a microwave oven until completely dissolved. After clarification, add 10 μL of SYBR Green I nucleic acid fluorescent tracer and vortex for 10 seconds to mix thoroughly. Cool to 50°C and pour into a gel casting mold. Let stand at room temperature for 30 minutes to allow the gel to solidify. Transfer the solidified gel horizontally into the electrophoresis tank and slowly add TAE running buffer along the tank wall until the seal layer reaches 1-2 mm. Add 7 μL of DNA molecular weight standard to the first well, and then load equal volumes of sample into subsequent wells in parallel. After sample addition, connect the electrophoresis instrument's positive and negative terminals and power supply, set the voltage to 120 V, and run for 20 minutes. After the gel electrophoresis is complete, remove the gel and inspect for bands using a gel imaging camera. Record the results.

[0046] The dead goose tissues were homogenized, frozen and thawed, and the total DNA and total RNA were extracted for PCR and RT-PCR. Figure 1 As shown, except for the specific band of about 489bp of GAstV, goose circovirus, avian adenovirus, Newcastle disease virus, goose parvovirus, Tembusu virus, avian influenza virus and avian reovirus did not amplify the target fragment. It was preliminarily determined that the gout in the farmer's geese was caused by GAstV infection.

[0047] 1.3 Virus isolation and culture 1.3.1 Virus culture in goose embryos Use the virus solution prepared in Section 1.1 to inoculate healthy 9-day-old goose embryos via the chorioallantoic cavity, inoculating 0.3 mL per embryo. Simultaneously, set up a control group where all conditions remain unchanged, except that the inoculum is replaced with an equal volume of PBS. During the first day of inoculation, observe the embryos every 12 hours for survival within 24 hours. Discard any embryos that die within 24 hours to prevent misdiagnosis due to bacterial infection. Keep the remaining embryos in a 37°C incubator, carefully controlling the temperature and humidity. Observe them daily. Collect any embryos that die within 7 days and place them in a -4°C refrigerator overnight for sacrifice. If they are still alive on day 7, place them in a -4°C refrigerator overnight for sacrifice.

[0048] Light an alcohol lamp in a clean workbench, open the air chamber of the goose embryo aseptically, observe the condition of the chorioallantoic membrane, collect the allantoic fluid, centrifuge at 5000r / min for 15min, carefully remove the embryo with tweezers, observe its pathological condition, cut off its head and limbs, grind the embryo and internal organs together, remove the supernatant after centrifugation, place in a -80℃ refrigerator and freeze and thaw repeatedly for 3 times, then continue centrifugation, mix the supernatant with the collected allantoic fluid, filter with a 0.22μm sterilizing filter, and continue to inoculate the next generation of goose embryos in the above manner, and inoculate for six consecutive generations.

[0049] The results showed that there were no deaths in the first generation of goose embryos, sporadic deaths occurred in the second generation, and from the fourth generation onwards, the mortality rate gradually stabilized, with deaths concentrated between 72 and 96 hours, and a mortality rate of 70%. Observation of the dead goose embryos and the control group revealed redness and swelling all over the body, with obvious hemorrhages. Furthermore, due to developmental stunting, the dead embryos had growth retardation, with noticeable thickening and edema of the chorioallantoic membrane. Necropsy revealed obvious hemorrhages in organs such as the liver and heart.

[0050] The specific primers for goose astrovirus detection in Table 2 were used to extract RNA from the mixed solution of allantoic fluid and ground embryos of six generations of goose embryos, and RT-PCR detection was performed. Specific bands were successfully amplified in each generation (such as Figure 2 ), which was consistent with the expected results, indicating that the virus strain can proliferate on goose embryos. Stable goose astrovirus can be obtained by inoculating goose embryos and harvesting their allantoic fluid and grinding the embryo body. It was named GastV-M705 strain and submitted for preservation.

[0051] 1.3.2 Virus culture on LMH cells 1.3.2.1 Cell recovery Remove the LMH cells stored in liquid nitrogen and place them on ice. Thaw them quickly in a 37°C water bath for about 1 minute. Add 7 ml of complete culture medium to a centrifuge tube. Add the thawed cells to the centrifuge tube and centrifuge at 3500 r / min for 5 minutes. Discard the supernatant, add 1 ml of culture medium to resuspend the cells, and then add them to a T25 cell culture flask. Add 4 ml of complete culture medium to mix well, and culture them in a 37°C, 5% CO2 incubator. Observe the cell status and density on the next day and change the medium if necessary.

[0052] 1.3.2.2 Cell passaging When the revived cells have grown to 90% of the bottom area of the culture flask, digest them. Aspirate the old culture medium and rinse the cells 2-3 times with an appropriate amount of PBS. Add 1 ml of 0.25% trypsin, ensuring that the trypsin covers the entire bottom of the flask. Observe the extent of digestion under an inverted microscope. When cell processes retract and intercellular spaces increase, add 4 ml of complete culture medium and shake the culture flask to terminate the trypsin digestion. Carefully pipette the adherent cells with a pipette to create a cell suspension, controlling the force to avoid creating large bubbles. Transfer the suspension to a centrifuge tube and centrifuge at 3000 rpm for 4 minutes. Discard the supernatant and resuspend the suspension in 3 ml of complete culture medium. Inoculate the tubes into pre-labeled culture flasks at a 1:3 ratio, bringing the volume up to 5 ml. Subculture the tubes in a CO2 incubator at 37°C, 5% CO2.

[0053] 1.3.2.3 Virus inoculation and collection After processing the allantoic fluid from which GAstV was isolated, the strain was tested on cells. When the passaged cells grew to approximately 70% of their normal density, the culture medium was discarded and the cells were rinsed twice with PBS to remove any residual culture medium to prevent the serum in the culture medium from affecting the adsorption of the virus. 1 ml of allantoic fluid was added and the cell bottle was gently shaken. The cells were placed in a 37°C, 5% CO2 incubator and incubated for 2 hours. Afterwards, the allantoic fluid was discarded and 5 ml of maintenance medium containing 2% FBS was added. The cells were cultured and observed daily. When the cells showed obvious pathological changes or after 96 hours, they were harvested and placed in a -80°C freezer, repeatedly frozen and thawed three times, and centrifuged at 4000 rpm for 15 minutes to remove cell debris. The supernatant was collected and filtered through a 0.22 μm sterilizing filter for subsequent passage.

[0054] Sixth-generation allantoic fluid, identified as positive for this strain, was used to inoculate LMH cells. While pathological changes were minimal in the first few generations, with only a small amount of cell shedding, significant cytopathic effects were observed by the fifth generation. The time it took for pathological changes to develop gradually stabilized with increasing passage number. Around 48 hours after inoculation, many LMH cells became rounded and shrunken, with poorer adherence. Over time, intercellular spaces enlarged, viral plaques appeared, light-blocking properties decreased, and cells gradually ruptured, shed, and died.

[0055] 1.3.2.4 Determination of virus TCID50 (1) When the passaged cells grow to 80-90% density, digest them into a 96-well cell culture plate, add 100 μL of cell solution to each well, and culture in a 37°C, 5% CO2 incubator for 24 hours.

[0056] (2) Dilute the virus solution. Prepare 12 sterile 1.5 mL centrifuge tubes (number them 1-12). Add 1000 μL of virus suspension to tube 1. Pre-load 900 μL of maintenance solution into tubes 2-11. Pipette 100 μL of virus solution from tube 1 to tube 2 and vortex to mix. Then, transfer 100 μL of virus solution from tube 2 to tube 3 and vortex to mix. Repeat this process until tube 11. After the final mixing, aspirate 100 μL and discard it. Tube 12 is set as a negative control, which is filled with 1000 μL of maintenance solution.

[0057] (3) Pour out the cell growth medium in the 96-well culture plate and rinse with PBS three times.

[0058] (4) Each column is treated as a dilution. 100 μL of each gradient virus dilution solution is added to each well. Column 12 is used as a negative control and an equal amount of maintenance solution is added. Place the wells in a 37°C, 5% CO2 incubator and incubate for 1 hour.

[0059] (5) Remove the virus dilution solution, add appropriate amount of PBS to rinse three times, add 100 μL of cell maintenance solution to each well, place in the incubator for culture, observe the cell pathological changes every day, record them, and finally calculate the virus half-cell infectious dose (TCID) according to the Reed-Muench method. 50 ).

[0060] The 5th generation of cytotoxic cells were selected and their TCID 50 The cytotoxicity of the fifth generation was calculated by the Reed-Muench two-way method. 50 =10 -5.45 / 0.1 mL (Table 4).

[0061] Table 4 TCID calculated by Reed-Muench method50 result

[0062] 1.3.3 Determination of the one-step virus growth curve Take the F11 generation virus liquid, and when the LMH cell density grows to about 90%, transfer it to a 6-well cell culture plate according to the method in 1.3.2.2, and wash it twice with PBS. Subsequently, inoculate the sample according to 10% of the total culture medium, and place it in an incubator at 37°C and 5% CO2 for incubation for 1 hour. Then, aspirate the virus liquid, add maintenance culture medium, and return the culture plate to the incubator for static culture. At different time points after inoculation (0h to 72h, sample every 12h), take out 0.5mL of supernatant culture medium and add an equal amount of maintenance medium to keep the volume of the culture medium unchanged. Subsequently, determine the TCID in the culture virus liquid at different time points according to the previously described method. 50 , according to the TCID at each time point 50 The titer values were used to draw the one-step growth curve of the virus.

[0063] The virus solution on the six-well cell culture plate was collected at 12h, 36h, 48h, 60h, and 72h after infection, and the TCID 50 The results showed that GAstV M705 strain can stably proliferate on LMH cells. The virus titer increased rapidly in 12-36h, reached the maximum in 48h, and then gradually decreased with the passage of time. 48h is also the best time to collect the virus. Figure 3 Middle A).

[0064] 1.3.4 Virus titer change curve GAstV was continuously passaged in cells, and the cytotoxicity of each generation was measured by TCID50 in 1.3.2.4. The cytotoxicity curve was then drawn. The results showed that the cytotoxicity of GAstV was stable at 10 6 / mL or above ( Figure 3 Middle B).

[0065] 1.3.5 Morphological observation of the virus Collect the cytotoxic cells with positive test results, purify the virus by differential centrifugation, and observe the morphology of the virus particles under a transmission electron microscope. The specific steps are as follows: Take about 10 ml of virus cell fluid and centrifuge it at 4°C, 9000 r / min for 40 min, collect the supernatant; centrifuge it at 4°C, 10000 r / min for 30 min, and retain the supernatant; ultracentrifuge the obtained supernatant at 4°C, 40000g for 4 hours, retain the precipitate, add electron microscopy fixative stored at 4°C to the precipitate, mix it gently by pipetting, and send the suspension to Chengdu Saiinster Biotechnology Co., Ltd. for negative staining with 1% phosphotungstic acid and then observe it by transmission electron microscopy. The results are as follows Figure 4 As shown, the virus particles are approximately spherical in shape, with a diameter of approximately 30 nm and no envelope, which is consistent with the morphology of astroviruses reported in the literature.

[0066] 1.3.6 Determination of the physical and chemical properties of the virus 1.3.6.1 Thermal sensitivity test The 11th-passage virus solution (1 mL / tube) obtained using the cell passaging method described in 1.3.2.2 was aliquoted into two parallel treatment systems: the experimental group was placed in a 60°C constant-temperature water bath for 30 minutes, while the control group was maintained in standard culture conditions (37°C) for simultaneous incubation. The TCID50 values of the two groups were then measured.

[0067] 1.3.6.2 Trypsin sensitivity test The 11th generation virus solution was aliquoted into two sets of centrifuge tubes (1 mL each). 5 μL of 1% trypsin solution was added to the experimental group and vortexed to a final concentration of 0.5% (v / v). The cells were then transferred to a 37°C constant temperature water bath and digested for 2 hours. The enzymatic reaction was terminated with an appropriate amount of PBS. In the control group, an equal volume of PBS was used instead of trypsin, and all other procedures were the same. The TCID values of the two groups were determined. 50 .

[0068] 1.3.6.3 Chloroform sensitivity test The 11th generation virus solution was aliquoted into two sets of centrifuge tubes in parallel. The experimental group was treated with chloroform (final concentration 4.8% v / v) and continuously mixed on a low-temperature shaker (4°C, 150 rpm) for 10 minutes. The upper aqueous phase was then collected by gradient centrifugation at 4°C (3000 rpm, 20 minutes). The control group was subjected to the same procedure using an equal volume of PBS instead of chloroform. The TCID values of the two groups were then determined. 50 .

[0069] 1.3.6.4 Acid resistance test The 11th generation virus solution was divided equally into two sets of centrifuge tubes (1 mL each). The experimental group was treated with a gradient acidification of 0.1 mol / L HCl to pH 3.0, maintained at 25°C for 2 hours, and then titrated with 5.6% NaHCO3 solution to a physiological pH of 7.0. The control group was treated with an equal volume of PBS instead of the acid treatment reagent, and the other operating parameters were kept the same. Finally, the TCID of the two groups was determined. 50 .

[0070] The results of the above four tests are as follows Figure 5 As shown, after the virus was treated with chloroform and trypsin, its toxicity was not significantly different from that of the control group, indicating that the virus has a certain resistance to organic lipid solvents and trypsin, and conventional organic solvents cannot completely inactivate it. After treatment at 60°C for 30 minutes, the virus toxicity was not different from that of the control group, indicating that the virus is not sensitive to heat and can continue to survive in the environment at high temperatures. In an acidic environment of pH 3.0, the virus toxicity decreased by approximately one order of magnitude, indicating a slight sensitivity to acidic environments.

[0071] Example 2:

[0072] Animal regression test, details are as follows: 2.1 Experimental groups and challenge Twenty one-day-old goslings were randomly divided into two groups and fed normally until they were three days old. The challenge test was conducted. The challenge group was injected intramuscularly with the sixth generation of cytotoxicity (for the acquisition of the sixth generation of cytotoxicity, refer to Section 1.3.2.2 Cell passage in Example 1) (approximately 10 5.8 TCID 50 ), the injection volume was 0.5 mL, and the control group was inoculated with the same amount of PBS. The goslings in each group were raised in separate cages, and the groups were shown in Table 5.

[0073] Table 5 Animal regression experiment groups

[0074] 2.2 Observation of clinical symptoms and death The changes of goslings in the challenge group were observed within 20 days. The body weight of each group was weighed and recorded every 4 days. The clinical symptoms and mortality of goslings in the challenge group were observed. Figure 6 As shown, no deaths occurred in the control group, who ate and drank normally throughout the entire process. However, some people in the challenge group began to experience loss of appetite and depression starting on day 3, and their weight was significantly lower than that of the control group starting on day 12. In the control group without the virus, no abnormalities or deaths were observed throughout the entire process.

[0075] 2.3 Anatomical lesion observation Goslings that became ill or died were promptly autopsied. Goslings that survived were humanely sacrificed on the last day of life. Pathological changes in the liver, kidneys, and other internal organs, as well as leg joints, were observed and recorded. Tissues including the heart, liver, spleen, kidney, brain, and pancreas were collected as specimens. Portions of the kidneys, liver, heart, and spleen were fixed in 4% paraformaldehyde buffer.

[0076] Of the 10 goslings exposed to the poison, six died starting on the fifth day. The dead goslings exhibited opisthotonos and tear marks in their eye sockets. Autopsies revealed urate deposits in the liver, heart, and kidneys. A white membrane covered the heart; diffuse white urate deposits were seen on the surface of the glandular stomach and sternal membrane; white urate deposits were present in the ureters; the kidneys were whitish and swollen, and there were dendritic hemorrhages under the skin. No gout was observed in the control group.

[0077] Example 3:

[0078] The whole genome sequencing and genetic evolution analysis of the GAstV M705 strain are as follows: 3.1 Primer design Six full-length amplification primers were designed and synthesized by Qingke Biotechnology Co., Ltd. The specific information of the designed primer sequences is shown in Table 6.

[0079] Table 6 Primers for PCR amplification of whole genome sequences

[0080] 3.2 Whole-genome PCR amplification The cDNA synthesized in Section 1.2.2 of Example 1 was used to amplify the full viral genome sequence one by one using the primers designed in Section 3.1. The reaction system is shown in Table 7. The reaction conditions are: 94°C pre-denaturation for 5 min; 94°C denaturation for 30 s, 58°C annealing for 30 s, 72°C extension for 30 s, 35 cycles; 72°C extension for 10 min; 4°C storage, ∞. The PCR results are shown in Table 7. Figure 7 As shown in the figure, the expected amplified fragment lengths of the six primer pairs were all above 1000 bp.

[0081] Table 7 Reaction system

[0082] 3.3 Recovery and purification of target genes After electrophoresis, the gel was transferred to a UV imaging system. A sterile scalpel blade was used under a UV hood to precisely excise the target band and exclude regions of nonspecific amplification. The gel fragments were transferred to pre-weighed 1.5 mL EP tubes, and the net weight of the gel pieces was determined using an analytical balance. The viral genomic fragments were purified using silica gel membrane adsorption, and the final product was sent to Qingke Biotech Co., Ltd. for sequencing.

[0083] 3.4 Whole-genome sequence assembly and evolutionary analysis The complete genomes of astroviruses from different clades were retrieved and their sequences downloaded through the NCBI GenBank database. Multiple sequence alignments were performed using Clustal W on the MEGA 11.0 platform, and a phylogenetic tree was constructed using the neighbor-joining method. Global sequence alignments were then performed using the DNASTAR Megalign module, and a nucleotide homology matrix was calculated. Finally, the IEDB analysis platform was used to predict antigenic epitopes in the ORF2 protein of the target strains.

[0084] The sequenced sequences were assembled and spliced to produce the complete genome sequence of the GAstV M705 strain. The assembled sequence revealed a full length of 7187 base pairs. Comparison with other GAstV strains uploaded to NCBI revealed a typical astrovirus structure, consisting of two non-coding regions and three open reading frames in the middle: 5'UTR-ORF1a-ORF1b-ORF2-3'UTR.

[0085] To further reveal and analyze the genetic lineage differentiation characteristics across species and virus subtypes, and to combine multiple sequence alignment to reveal the correlation between high-frequency gene recombination events and cross-species adaptive evolution, we selected other mammalian and avian astrovirus reference strains and downloaded their full genome sequences, including human astrovirus (HAstV), porcine astrovirus (PAstV), bovine astrovirus (BAstV), bat astrovirus (PaAstV), duck astrovirus (DAstV), chicken astrovirus (CAstV), turkey astrovirus (TAstV), and goose astrovirus (GAstV). BLAST comparison of the spliced virus strain with these strain sequences revealed that the M705 strain had the strongest homology with the G538 strain among all goose astrovirus strains, reaching 99.46%. The phylogenetic evolutionary tree of the whole genome, ORF1a, ORF1b, and ORF2 was drawn using MEGA 11.0 software ( Figure 8 ), MegAlign software was used to create a nucleotide homology analysis comparison diagram of the whole genome, ORF1a, ORF1b, and ORF2 ( Figures 9 to 12 ).

[0086] According to existing literature reports and the analysis of the phylogenetic evolutionary tree of the whole genome, goose astrovirus is divided into two main lineages, showing two different branches in the evolutionary tree, and the two branches are very far apart in relationship, that is, there are two genotypes, type I goose astrovirus (GAstV-1) represented by FLX, TZ03, etc. and type II goose astrovirus (GAstV-2) represented by SDPY, JSSQ, etc., while duck astrovirus, chicken astrovirus, turkey astrovirus, etc. are all on the same branch, which is different from goose astrovirus, suggesting that goose astrovirus may have evolved from other viruses.

[0087] Since the outbreak of goose astrovirus in my country, the outbreak has been more serious in Guangdong, Anhui, Jiangsu and other places. As time goes by, there is a trend of spreading from coastal areas to inland areas and from eastern areas to western areas. The whole genome sequence of the M705 strain isolated by the present invention is in different small branches of the same branch as the G548 and G538 strains in the phylogenetic tree, indicating that it may have evolved from these two strains. According to nucleotide homology analysis, it has the highest homology with the G538 strain, reaching 99.5%, and with the G548 strain is 99.4%. The homology with other GAstV-2 strains is between 97.8% and 99.4%, indicating that the M705 strain belongs to the typical GAstV-2, while it is obviously not on the same branch as the representative GAstV-1 strains FLX, AHDY and TZ03 strains, indicating that their genetic relationship is very far apart. The ORF1a and ORF2 sequences were also in different subbranches of the same branch as the G548 and G538 strains, with ORF1a homologies of 99.7% and 99.5%, respectively, and ORF2 homologies of 99.2%. ORF1b was in the same branch as the XSSH strain, with a homology of 99.2%.

[0088] Example 4:

[0089] The preparation and immunogenicity analysis of inactivated virus antigens are as follows: 4.1 Inactivation of viral fluid The 11th generation cell virus liquid was selected as the seed batch for antigen production, and its TCID 50 =10 -6.26 / 0.1mL, and inactivated with formaldehyde. To investigate the inactivation effects of different formaldehyde concentrations and durations, formaldehyde solutions were added at final concentrations of 0.1%, 0.2%, and 0.3%, with treatments for 12, 24, and 36 hours, respectively. The specific groupings are shown in Table 8. The specific procedure was as follows: add formaldehyde solutions at 0.1%, 0.2%, and 0.3% of the volume of the virus solution, vortex and mix thoroughly in a centrifuge, place in a 37°C shaker, and shake every 6 hours. A 1ml sample was collected from each volume gradient every 12, 24, and 36 hours for inactivation testing. The sample was then inoculated into LMH cells and observed for 72-120 hours to detect cell pathology. Cell culture fluid was collected for RNA extraction and RT-PCR (see Section 1.2.4 of Example 1) to observe the appearance of specific bands. The sample was blindly passaged for three consecutive generations, with the same procedure repeated for each generation. The results are shown in Table 9.

[0090] Table 8 Optimization of antigen inactivation conditions

[0091] Table 9 Formaldehyde concentration and inactivation time

[0092] The results in Table 9 show that all virus tests were positive in the groups with final formaldehyde concentrations of 0.1% to 0.2%, while negative results were observed after 24 hours at a final formaldehyde concentration of 0.3%, indicating that the antigen was completely inactivated. This indicates that formaldehyde concentration has a certain impact on the inactivation effect. Considering the time cost, a 0.3% formaldehyde solution was used for inactivation for 24 hours.

[0093] 4.2 Preparation of oil phase After premixing medicinal white oil (No. 10) and emulsifier Span-80 in a volume ratio of 94:6, 2% (w / w) aluminum stearate was added as a stabilizer. The mixture was stirred by magnetic stirring at a gradient temperature of 80-120°C until a homogeneous transparent phase was formed with no granular impurities visible to the naked eye. The mixture was then sterilized by high pressure at 121°C and finally stored in a brown glass container in a dark place until used.

[0094] 4.3 Preparation of aqueous phase The inactivated antigen solution (prepared in Section 4.1) was mixed with sterile Tween-80 at a volume ratio of 39:1, placed in a homogenizer to mix, and then centrifuged at 6000 rpm for 20 min until no stratification was visible to the naked eye.

[0095] 4.4 Emulsification Measure the oil phase and water phase in a volume ratio of 3:2. Add the oil phase to a sterile beaker. Place the beaker under the homogenizer and adjust the homogenizer rod so that it is at the bottom of the beaker. Stir to thoroughly mix the two phases, creating a vortex without generating a large number of bubbles. Initially, adjust the gear to 3 and slowly add the water phase. Once the water phase is completely added, adjust the gear to 6 and homogenize for 8 minutes, with a 3-minute interval. Repeat three times to fully blend the two phases.

[0096] 4.5 Quality Inspection of Inactivated Viruses 4.5.1 Dosage form inspection The vaccine prepared in Section 4.4 was dripped vertically onto the surface of a plate containing ultrapure water. The vaccine's diffusion was observed after the droplets were dropped to determine the dosage form. If the first droplet diffused but subsequent droplets did not disperse, it was an oil-in-water type. If the first droplet diffused immediately, it was an oil-in-water type. The test results showed that the first droplet was diffusely distributed on the water surface, while the second and subsequent drops appeared as regular, round, oil-like droplets floating on the cold water surface and did not disperse even with slight shaking. Therefore, the inactivated virus in this example was an oil-in-water type, meeting relevant standards.

[0097] 4.5.2 Stability Test Aliquot the inactivated vaccine sample (4 mL x 2) into two 15 mL conical centrifuge tubes and centrifuge at 3000 rpm for 15 minutes at 4°C. Observe the vaccine for signs of demulsification or stratification. If no demulsification or demulsification occurs, the centrifugal stability is good. The test results indicate that the reagent exhibits no signs of demulsification or stratification, indicating good stability.

[0098] 4.5.3 Viscosity test Using a 1mL standard pipette (1.2mm inner diameter), vertically release 0.4mL of vaccine sample at room temperature, allowing it to flow freely. A timer was used to measure the flow time three times in a row, and the average time was calculated. The test results showed three times of 5.3s, 5.2s, and 5.5s, respectively, with an average of 5.3s, which is within 2s-8s, indicating that the prepared reagent meets the viscosity requirements.

[0099] 4.5.4 Sterility testing To evaluate the sterility of the vaccine, the prepared oil-emulsion inactivated vaccine was inoculated into LB, PDA, and MacConkey medium to monitor for potential bacterial and fungal contamination. The test results showed no microbial growth after 120 hours in an incubator, confirming that the inactivated virus prepared in this example passed the sterility test.

[0100] 4.5.5 Vaccine safety testing Twenty one-day-old goslings were purchased and randomly divided into two groups, 10 in each of the experimental group and the control group. The experimental group was subcutaneously injected with 1.0 mL of inactivated vaccine on the dorsal side of the neck, and the control group was injected with an equal volume of sterile white oil adjuvant at the same location. After vaccination, the test animals were observed every day for their appetite, mental state, and whether there were any related adverse reactions. After 14 days, the goslings were autopsied to observe the absorption at the injection site. The test results showed that the test group had mild adverse reactions within 2 days after vaccination, but gradually returned to normal afterwards, and no deaths occurred. After a 14-day observation period, the test group and the control group were in good mental state, with normal growth and development. After autopsy, the test group absorbed the injection site well, without tissue necrosis or inflammatory reaction, and no pathological changes occurred. The above results indicate that the vaccine prepared in this embodiment has reliable safety.

[0101] 4.6 Inactivated virus immune effect testing 4.6.1 Inactivated virus immunization Sixty one-day-old goslings were purchased and raised for two days until stable. At three days of age, they were randomly divided into five groups of 12 goslings each, with footmarks applied to each group. Each group received different doses of the vaccine, while one group served as a negative control. Serum was collected from the jugular vein of five goslings randomly selected from each group at 7, 14, 21, 28, and 35 days after immunization for immunogenicity studies. Specific groupings are shown in Table 10.

[0102] Table 10 Immunity test

[0103] 4.6.2 Indirect ELISA for IgG Levels in Serum After immunizing goslings with inactivated virus, in order to detect whether specific antibodies are produced in the goslings and the level of specific antibodies, ELISA is used to detect the level of specific antibodies in the collected gosling serum. The specific operation is as follows.

[0104] (1) Coating. After purifying the viral allantoic fluid, dilute it appropriately with coating buffer (0.05 mol / L carbonate buffer, pH = 9.6) and coat the ELISA plate at 100 μL / well. Cover the plate and place it in a 4°C refrigerator for overnight coating.

[0105] (2) Washing. Use PBST to wash the wells. Add 200 μL of PBST to each well and place the well in a plate shaker. Wash four times, 5 minutes each time. After the last wash, pat dry the remaining liquid in the air.

[0106] (3) Blocking. Prepare 5% BSA as blocking solution and add 300 μL of blocking solution to each well. Add slowly to prevent bubbles from forming. Place in a 37°C oven for 1 hour.

[0107] (4) Washing. After blocking, wash the wells with PBST. Add 200 μL of PBST to each well and place the well in a plate shaker. Wash four times, each time for 5 minutes. After the last wash, pat dry the remaining liquid in the air.

[0108] (5) Incubate with primary antibody. Add the prepared serum to be tested to the ELISA plate at an appropriate dilution at 100 μL / well. Set up 4 replicate wells for each sample, cover the plate, and incubate in a 37°C oven for 2 h.

[0109] (6) Washing. After incubation and blocking, wash the wells with PBST. Add 200 μL of PBST to each well and place the well in a plate shaker. Wash four times, each time for 5 minutes. After the last wash, pat dry the remaining liquid in the air.

[0110] (7) Incubate with secondary antibody. Prepare 0.5% BSA as the secondary antibody diluent, dilute HRP-labeled goat anti-goose IgG at 1:4000, add 100 μL to each well, and incubate in a 37°C oven for 1 hour, protecting from light.

[0111] (8) Washing. After incubation and blocking, wash the wells with PBST. Add 200 μL of PBST to each well and place the well in a plate shaker. Wash four times, 5 minutes each time. After the last wash, pat dry the remaining liquid in the well.

[0112] (9) Color development. Add 100 μL / well of TMB color development solution and place in a 37°C oven in the dark for 20 minutes (or at room temperature in the dark for 30 minutes).

[0113] (10) Determine OD450. Prepare 2M concentrated sulfuric acid and add 100 μL to each well to stop color development. Measure the OD450 value using a microplate reader within half an hour.

[0114] Test results such as Figure 12 As shown, specific antibodies were detected in all immunization groups 7 days after immunization, with a significant increase compared to the control group. Other immunization groups showed significant differences from the 0.2 ml immunization group, but no significant differences were observed between the groups. Antibody levels in all immunization groups gradually increased from 14 days after immunization, reaching a peak on 21 days. The 0.2 ml immunization group showed the smallest increase, while the 1 ml immunization group reached the highest antibody level (OD value approximately 1.43), which was statistically significant compared to the control group. After 21 days, antibody levels in all immunization groups declined, but by 35 days, antibodies in the high-dose group were still maintained at a certain level (OD value around 0.8-1). Specific antibody levels in the control group were low throughout the entire process, with almost no specific antibodies produced.

[0115] 4.6.3 Detection of serum neutralizing antibody levels We used serum neutralization tests to analyze whether GAstV neutralizing antibodies were produced in the goslings after immunization, and used LMH cells for detection. The specific procedures are as follows.

[0116] (1) Inactivate the serum to be tested in a constant temperature water bath at 56°C for 30 minutes.

[0117] (2) The inactivated serum to be tested was diluted 2-fold using PBS solution (1:2, 1:4, 1:8, 1:16, 1:32, 1:64...).

[0118] (3) Dilute the virus with DMEM to 200 TCID50 / 0.1 mL, mix the diluted virus with the diluted serum to be tested in a 1:1 ratio, and incubate in a 5% CO2 incubator at 37°C for 2 h, mixing every 30 min.

[0119] (4) Add the incubated mixture to the pre-prepared 96-well LMH cell culture plate at 100 μL / well and incubate in a % CO2 incubator for 2 h.

[0120] (5) After the infection, discard the liquid and wash twice with PBS solution. Add 100 μL of cell maintenance solution to each well and culture in a 5% CO2 incubator at 37°C. Set up a blank control group, a positive serum control group, and a negative serum control group, and observe and record the number of cytopathic wells.

[0121] (6) Calculate the neutralizing antibody titer of the serum to be tested according to the Reed-Muench method.

[0122] Test results such as Figure 13 and Figure 14 As shown, no neutralizing antibodies were produced in the negative control group during the entire period. After immunization of goslings, the production of neutralizing antibodies was detected in all immunized groups, which gradually increased from the 7th day, and then the antibody level increased with time within 14 days. From the results, the growth trend of all groups was roughly similar, reaching the highest on the 21st day. At this time, the average antibody titers of each immunized group were 5.90log2, 6.04log2, 7.23log2, and 9.01log2, respectively. The titer of the 1ml immunized group was significantly higher than that of other immunized groups. Subsequently, the neutralizing antibody titer was detected until 35 days, and the neutralizing antibody titer was maintained at a high level. The neutralizing antibody titers of the three high-dose immunized groups were all higher than 6log2. The above results show that the vaccine can reach a high concentration level in the early growth of goslings, which can play a certain role in preventing GAstV.

[0123] 4.6.7 Challenge protection test At 35 days of age, goslings in each group were challenged with the virus via intramuscular injection into their legs. Each gosling was injected with 0.5 ml of GAstV M705 strain cytotoxin (106.25 TCID50 / 0.1 mL) and housed in separate groups. The goslings were housed for 10 consecutive days, during which their feeding habits, fecal status, and mental state were observed daily. Anal swabs were collected from six randomly selected goslings in each group 3, 5, 7, and 10 days after challenge. RNA was extracted, and RT-PCR was used to analyze the virus excretion. The results are summarized below: (1) Clinical symptoms and death During the 10-day observation period following the challenge, the vaccinated group experienced a brief decrease in feed intake and depression, but their appetite and behavior returned to normal after the challenge. A small number of deaths occurred in the control group after the challenge, but no typical clinical symptoms were observed.

[0124] The clinical symptoms and mortality of ducklings were observed and recorded within 10 days. The results are shown in Table 11. In the negative control group, 5 ducklings died, with a survival rate of 58%; in the 0.2 ml group, 3 ducklings died, with a survival rate of 75%; no ducklings died in the other three groups, with a protection rate of 100%.

[0125] Table 11 Gosling survival rate

[0126] (2) Detoxification results Cloacal swabs were randomly collected from each group on days 3, 5, 7, and 10 after challenge, and RNA was extracted and tested for GAstV by RT-PCR. The results (Table 12) show that all negative control groups tested positive for nucleic acid within 10 days after challenge, indicating a prolonged period of virus shedding. Positive samples were detected in the 0.2ml immunization group from days 3 to 10, with fewer positive samples detected on days 3 and 5 and more on day 7. Positive samples were occasionally detected in the 0.5ml immunization group, but were no longer detected after day 7. No positive samples were detected in the 0.8ml and 1ml immunization groups throughout the entire process. Analysis of virus shedding in goslings after challenge revealed that complete protection was not achieved with 0.2ml of the vaccine, with frequent positive samples detected, indicating that the virus was still in the body. However, no deaths occurred with 0.5ml or higher doses, and while viral genes were detected, the virus quickly resolved, indicating that this dose provides adequate protection.

[0127] Table 12 Gosling detoxification test results

[0128] In summary, the present invention uses goose embryos and LMH cells to isolate a goose astrovirus GAstV M705 strain from clinical disease materials. The strain can produce cytopathic effects when continuously subcultured on LMH cells, and the virus titer is stable at 10 6.0 Phylogenetic tree and homology showed that the M705 strain belongs to genotype II with most GAstV strains, and its homology with the typical type I strains FLX and AHDY is only 57.5%.

[0129] The present invention further utilizes the GAstV M705 strain to prepare an inactivated vaccine. This vaccine exhibits stable properties, good safety, and produces high levels of antibodies after immunization of goslings. It also features a simple preparation process and can subsequently be mass-produced using suspension culture flasks for cell culture according to industrial needs. This vaccine is expected to provide a reference for the development of GAstV-related vaccine products in the domestic market, offering new insights into GAstV prevention and control, and becoming a potential vaccine candidate.

[0130] Although the embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations may be made to these embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents. The embodiments described above merely illustrate several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they cannot be construed as limiting the scope of the patent of the present invention. It should be pointed out that, without departing from the concept of the present invention, a number of variations and improvements may be made by those skilled in the art, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent of the present invention shall be based on the appended claims.

Claims

1. A goose astrovirus, characterized in that The goose astrovirus is named goose astrovirus GAstV-M705, and its taxonomic name is Avastrovirus-Goose astrovirus , deposited in the China Center for Type Culture Collection, the deposit number is CCTCC NO: V202521, and the deposit date is April 29, 2025.

2. Use of the goose astrovirus according to claim 1 in the preparation of a vaccine for preventing and treating goose astrovirus.

3. An inactivated vaccine for preventing and treating goose astrovirus, characterized in that: The inactivated vaccine contains the goose astrovirus according to claim 1.

4. The inactivated vaccine according to claim 3, characterized in that The goose astrovirus adopts cell virus liquid of the fifth passage or above.

5. The inactivated vaccine according to claim 4, characterized in that The goose astrovirus adopts cell virus liquid of the sixth passage or above.

6. The inactivated vaccine according to claim 4 or 5, characterized in that TCID of the cell virus solution 50 >10 -6 / 0.1mL.

7. The method for preparing the inactivated vaccine according to any one of claims 3 to 6, characterized in that: The steps include: Step 1, inactivating the goose astrovirus liquid to obtain an antigen liquid; Step 2, preparing an oil phase and an aqueous phase respectively, wherein the preparation of the oil phase comprises: premixing a medicinal white oil with an emulsifier, adding a stabilizer, heating the mixture to a temperature of 80-120° C. in a gradient manner, mixing the mixture until a homogeneous transparent phase is formed, and then sterilizing the mixture to obtain the oil phase; The preparation of the aqueous phase comprises: mixing the antigen solution with sterile Tween-80, separating the insoluble matter, and obtaining the aqueous phase; Step 3: mixing the oil phase and the water phase in a volume ratio of 2 to 3:1 to obtain the inactivated vaccine.

8. The preparation method according to claim 7, characterized in that In step 1, the inactivation method includes: using formaldehyde as an inactivator, and the inactivation time is 20 to 40 hours; preferably, the amount of formaldehyde added is 0.3% of the volume of the virus liquid, and the inactivation time is 20 to 24 hours.

9. The preparation method according to claim 7, characterized in that In step 2, the medicinal white oil and the emulsifier are premixed in a volume ratio of 93-97:3-7, and the amount of the stabilizer added is 1-4% of the total mass of the oil phase; and / or, the antigen solution and sterile Tween-80 are mixed in a ratio of 30-40:1-2; and / or, the insoluble matter is separated by centrifugation. Preferably, the control parameters of the centrifugation include: 4000-8000 rpm, and a centrifugation time of 10-20 min.

10. The preparation method according to claim 7, characterized in that In step 3, the method of mixing the oil phase and the water phase includes: adding the water phase to the oil phase at a mixing speed of 400-600 rpm, adjusting the mixing speed to 800-1200 rpm after the addition, continuing the homogenization for 6-10 minutes, taking an interval of 3 minutes, and repeating 3 times in a row to fully blend the two phases.

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