A goose-origin Muscovy duck reovirus, inactivated vaccine and its application

Abstract

The present invention discloses a goose-origin Muscovy duck reovirus, an inactivated vaccine thereof and its application, belonging to the field of isolation and application of avian reovirus strains. The virus was deposited at the China Center for Type Culture Collection on October 30, 2024, and its deposit number is: CCTCC NO: V202491. The goose-origin Muscovy duck reovirus provided by the present invention has good immunogenicity against goose-origin Muscovy duck reovirus with main symptoms of a large number of white necrotic spots in tissues and organs such as the liver and spleen and joint cavity hemorrhage. The inactivated vaccine prepared with this goose-origin Muscovy duck reovirus strain has good safety, with a protection rate of 100%, and can provide complete protection against newly isolated goose-origin Muscovy duck reovirus variant strains.

Description

Technical Field

[0001] The present invention relates to the field of isolation and application of avian reovirus strains, and particularly to a goose-origin Muscovy duck reovirus, an inactivated vaccine thereof, and applications thereof. Background Art

[0002] Disclosing the information of this background art section is only intended to enhance the overall understanding of the present invention, and it is not necessarily regarded as an admission or an implication in any form that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] Avian orthoreovirus (ARV) is a non-enveloped, icosahedral, double-layered capsid virus with a segmented double-stranded RNA and a diameter of 50 - 80 nm. ARV belongs to the family Reoviridae, subfamily Spinareovirinae, genus Orthoreovirus. ARV can cause various diseases in multiple avian species, and its clinical manifestations vary depending on the virus strain, virulence, or infected host, seriously endangering the development of the poultry industry in China.

[0004] Given that it is a newly emerging disease, there is currently a lack of in-depth research on its epidemiology and pathogenic mechanism, etc., and there are no effective vaccines or drugs for prevention and treatment. Therefore, starting from the pathogen, further studying the molecular pathogenic mechanism and virus mutation mechanism, and developing corresponding vaccines are the keys to preventing and controlling this infectious disease. Therefore, there is an urgent need to develop an inactivated vaccine that is safe and highly effective in preventing this goose-origin Muscovy duck reovirus. Compared with live attenuated vaccines, inactivated vaccines are relatively safe, simple to produce, and easy to store, and are also of great significance for the prevention and control of this disease and the healthy development of the goose industry. Summary of the Invention

[0005] In view of the above-mentioned prior art, the present invention isolates a strain of Muscovy duck reovirus that causes "flower liver disease" in goslings from diseased goslings, and investigates the cell tropism, pathogenicity, and immunogenicity of this goose-origin Muscovy duck reovirus, providing a guarantee for the prevention and treatment of newly emerging infectious diseases mainly characterized by "flower liver disease" and severe bleeding in joint cavities.

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

[0007] In the first aspect of the present invention, a goose-origin Muscovy duck reovirus is provided. The virus was deposited at the China Center for Type Culture Collection (abbreviated as CCTCC) on October 30, 2024. The deposit name is: Muscovy duck reovirus MDRV-JS001, and the taxonomic name is: Muscovy duck reovirus ( Muscovy Duck Reovirus ), and the deposit address is: Wuhan University, Wuhan, China, and the deposit number is: CCTCC NO: V202491.

[0008] The Muscovy duck reovirus of goose origin provided by the present invention is a mutant strain of the existing avian reovirus. Infected poultry will show a large number of white necrotic spots in the liver, spleen or other tissues and organs, and will also show symptoms such as listlessness, loss of appetite, movement disorders, and excretion of yellowish-green loose stools. In addition, the infected poultry will also show swollen joints, and severe bleeding in the joint cavity can be seen during autopsy, and the above-mentioned diseases have not occurred in the infection of Muscovy duck reovirus in the past.

[0009] Preferably, the poultry is a gosling.

[0010] In the second aspect of the present invention, there is provided the use of the above-mentioned Muscovy duck reovirus of goose origin in the preparation of a vaccine for preventing and treating "flower liver disease" and joint cavity bleeding in goslings.

[0011] The "flower liver disease" refers to a large number of white necrotic spots appearing in the liver, spleen or other tissues and organs of goslings caused by the infection of Muscovy duck reovirus with the preservation number: CCTCC NO: V202491.

[0012] Preferably, the vaccine is any one of an inactivated vaccine, a live attenuated vaccine, a subunit vaccine or a genetic engineering vaccine.

[0013] By investigating the cell tropism and immunogenicity of the Muscovy duck reovirus of goose origin provided by the present invention, it is found that the strain has a wide cell tropism, can proliferate on LMH cells, and can proliferate in large quantities in a short time, which is very suitable for the production of high-quality inactivated vaccines with high antigen titers, and has good immunogenicity against newly emerging infectious diseases with "flower liver disease" and joint cavity bleeding in goslings as the main symptoms. Therefore, the Muscovy duck reovirus of goose origin provided by the present invention can be used as an excellent vaccine strain for the preparation of vaccines.

[0014] The term "cell tropism" refers to the ability to infect and / or replicate in one or more cell lines or tissue cells.

[0015] In the third aspect of the present invention, there is provided an inactivated vaccine for preventing and treating "flower liver disease" and joint cavity bleeding in geese. The inactivated vaccine contains an inactivated Muscovy duck reovirus solution of goose origin in a prophylactically or therapeutically effective amount; the preservation number of the Muscovy duck reovirus of goose origin is CCTCC NO: V202491.

[0016] The preparation method of the inactivated Muscovy duck reovirus solution of goose origin includes the following steps:

[0017] (1) Inoculate the Muscovy duck reovirus strain of goose origin with the preservation number CCTCC NO: V202491 into LMH cells. After proliferating and culturing the Muscovy duck reovirus of goose origin, break the cells by freeze-thawing, collect the supernatant, and purify it to obtain the Muscovy duck reovirus solution of goose origin;

[0018] (2) Add formaldehyde with a final concentration of 0.2% to the goose-origin Muscovy duck reovirus solution obtained in step (1), stir and inactivate at 37 °C for 16 h to obtain an inactivated goose-origin Muscovy duck reovirus solution.

[0019] The virus titer of the goose-origin Muscovy duck reovirus solution is 10 -6.2 TCID 50 / 0.1 mL.

[0020] Preferably, the inactivated vaccine further contains a pharmaceutically acceptable adjuvant.

[0021] The preparation method of the inactivated vaccine includes the following steps:

[0022] Take the inactivated goose-origin Muscovy duck reovirus solution, add Tween-80 and mix as the aqueous phase, take the mixture of white oil, aluminum stearate and Span-80 as the oil phase, mix the oil phase and the aqueous phase evenly at a ratio of 2:1, and emulsify to obtain an inactivated vaccine for preventing goose-origin Muscovy duck reovirus.

[0023] The beneficial effects of the present invention:

[0024] 1. A goose-origin Muscovy duck reovirus MDRV-JS001 is isolated and obtained in the present invention, which has the ability of cross-species transmission and can infect goslings. After being infected with this strain, the goslings will show the phenomenon of swollen joint cavities. During the necropsy of the diseased goslings, it is found that the infected goslings show symptoms mainly characterized by "flower liver disease" and joint cavity bleeding. And this strain has a wide range of cell tropism and can proliferate on LMH cells, which is very suitable for the production of high-quality inactivated vaccines with high antigen titers; it has good immunogenicity against newly emerging infectious diseases mainly characterized by "flower liver disease" and joint cavity bleeding in goslings. It can be used as an excellent vaccine strain for vaccine preparation.

[0025] (2) Through the analysis of the σC gene sequence of this strain, it is found that the nucleotide and amino acid homologies of the σC gene sequence of the MDRV-JS001 isolate with the classical Muscovy duck reovirus ZJ2000M strain are both above 95%. It has a relatively distant genetic relationship with other waterfowl reovirus isolates and is in different genetic evolution branches. The inactivated vaccine prepared with the goose-origin Muscovy duck reovirus strain with the preservation number of CCTCC NO: V202491 has good safety, and the protection rate reaches 100%. Moreover, the inactivated vaccine of the present invention can quickly reach a high concentration of antibody level in a short time, and has a long immune persistence period, which can provide rapid and long-term immune protection for goslings. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is a pathological change diagram of virus cultured in LMH cells; among them Figure 1A in the figure is a picture of goose-derived Muscovy duck reovirus JS001 inoculated into LMH cells 48 hours later. Figure 1 B in the figure is a picture of control cells that were not inoculated with the virus.

[0027] Figure 2 PCR identification results; in the figure, lane 1 is duck reovirus, lane 2 is duck Tembusu virus, lane 3 is duck astrovirus, lane 4 is duck circovirus, lane 5 is duck plague virus, lane 6 is duck hepatitis A virus, lane 7 is blank control, lane 8 is goose-derived Muscovy duck reovirus, and M is a 2000bp Marker.

[0028] Figure 3 This is the growth curve determination of goose-derived Muscovy duck reovirus JS001. DETAILED DESCRIPTION

[0029] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0030] The following detailed description is for illustrative purposes only and is intended to provide further explanation of the present application, rather than to limit the scope of the present invention.

[0031] RNA extraction kit was purchased from Beijing Kangwei Century Co., Ltd., ordinary agarose gel recovery kit was purchased from Beijing Quanshijin Company, reverse transcription kit was purchased from Bao Biotechnology (Dalian) Co., Ltd., 2×Es Taq MasterMix was purchased from Beijing Kangwei Century Co., Ltd., and DL2000 Marker was purchased from Bao Biotechnology (Dalian) Co., Ltd.

[0032] Example 1: Isolation and identification of goose-derived Muscovy duck reovirus

[0033] 1. Epidemiological survey:

[0034] Since March 2021, an infectious disease characterized by paralysis and a large number of white necrotic spots in tissues and organs such as the liver and spleen has broken out in goslings in Jiangsu, Liaoning and Henan provinces of my country. The disease mainly occurs in goslings aged 7 to 35 days. The diseased geese have a large number of white necrotic spots in all tissues and organs of the body. The diseased goslings also have clinical symptoms of swollen joints, and the mortality rate can be as high as 80%. The autopsy of the dead goslings showed the following main pathological changes: enlarged liver and spleen, with a large number of white necrotic spots on the surface, and severe bleeding in the joint cavity.

[0035] 2. Sample collection and processing:

[0036] Collect the liver tissues of diseased goslings, add sterile PBS, grind them into homogenate, freeze-thaw three times repeatedly, centrifuge to obtain the supernatant. After the supernatant is filtered and sterilized through a 0.22 μm filter, inoculate it onto LMH cells with good growth status, adsorb in a cell incubator for 30 min, discard the sample treatment solution, and add 2% cell maintenance solution to continue the culture; set a negative control with sterile physiological saline and incubate at 37°C. Observe and record the cell status daily. When the cell fusion phenomenon reaches 80%, collect the virus solution. Perform subsequent detections by blind passage three generations in the same method. After three generations of subculture, the degree of cell fusion tends to be stable, and typical cytopathic effects ( Figure 1 A in Figure 1 ) can appear in 80% of the cells 48 h after inoculation, and no lesions appear in the negative control ( B in

[0037]

[0038] 3. RT-PCR identification:

[0039] (1) RNA extraction: Freeze-thaw the collected cell virus solution repeatedly, centrifuge to obtain the supernatant, and extract the viral RNA according to the requirements of the RNA extraction kit instructions, and store it at -20°C for later use.

[0040] (2) Reverse transcription to obtain cDNA: The reverse transcription kit used is PrimeScript™ RT Master Mix with the catalog number RR036A from Takara Bio (Dalian) Co., Ltd. In a 200 μL PCR reaction tube, add 5×PrimeScript RT Master Mix × 2 μL and RNA extracted from the virus solution ~2 μL in sequence, and use RNase Free dH2O to make up to a 10 μl system. Place it in a PCR instrument for reaction, and the reaction conditions are 37°C for 15 min; 85°C for 5 s, and then store at 4°C.

[0041] Muscovy duck reovirus belongs to the genus Orthoreovirus in the family Reoviridae. The σC protein is a very important structural protein of Orthoreovirus and plays an important role in the pathogenic process of the virus. In addition, the σC protein-encoding gene is the gene fragment most prone to mutation in the genome. Therefore, in this study, a pair of specific primers for amplifying a 1002 bp fragment was designed according to the Muscovy duck reovirus σC gene sequence in the gene sequence database Genbank established by the National Center for Biotechnology Information in the United States and synthesized by Beijing Tsingke New Industry Biotechnology Co., Ltd.

[0042] Forward primer: 5′- CCGATTGGGCCGACATCTCAT-3′ (SEQ ID NO.1);

[0043] Downstream primer: 5′- ACTACCTCAAGTGGTCGCAA-3′ (SEQ ID NO.2).

[0044] Amplification was carried out using a 20 μL system: template cDNA × 2 μL, upstream and downstream primers each × 1 μL, 2×Es TaqMasterMix × 10 μL, and supplemented with ddH2O to a 20 μL system. After mixing and briefly centrifuging, it was placed in a PCR instrument for reaction. The reaction conditions were 95°C for 5 min, followed by 30 cycles of 95°C for 45 s, 52°C for 30 s, 72°C for 25 s, and then 72°C for 10 min, and stored at 4°C for later use.

[0045] Meanwhile, conventional duck - derived viruses were detected using reported specific primers, including: Duck reovirus (DRV), Duck Tembusu virus (TMUV), Duck astrovirus (DAStV), Duck circovirus (DuCV), Duck plague virus (DPV), and Duck hepatitis A virus (DHAV).

[0046] (4) Agarose gel electrophoresis:

[0047] The PCR amplification products were detected by 1% agarose gel electrophoresis. The results showed that specific bands corresponding to the expected size of 1002 bp appeared after electrophoresis of the products amplified by the primers of SEQ ID NO.1 and SEQ ID NO.2 in 1% agarose gel, indicating the presence of Muscovy duck reovirus of goose origin in the cell culture of the isolate ( Figure 2 ), named strain JS001. In addition, the conventional duck - derived virus detection of the isolate, including DRV, TMUV, DAStV, DuCV, DPV, DHAV, showed negative results, and no other virus contamination was detected.

[0048] 4. Sequencing and sequence analysis:

[0049] After gel extraction of the amplification products, they were sent to Beijing Tsingke New Industry Biotechnology Co., Ltd. for sequencing. After splicing the sequencing results, they were compared with Blast, and homology analysis was carried out with other waterfowl reoviruses at the same time.

[0050] The results showed that the σC gene sequence of this strain was most closely related to the classical Muscovy duck reovirus strain ZJ2000M, with nucleotide and amino acid homologies both above 95%. It was distantly related to other waterfowl reovirus isolates and was in different genetic evolution branches (Table 1). Thus, it can be determined that it is a Muscovy duck reovirus of goose origin.

[0051] Table 1: Nucleotide and amino acid homology analysis of the σC gene of MDRV - JS001 isolate and waterfowl reoviruses

[0052]

[0053] This strain was named Muscovy duck reovirus MDRV-JS001. And this strain was deposited, and the deposit information is as follows:

[0054] Name of the strain: Muscovy duck reovirus MDRV-JS001

[0055] Depositary institution: China Center for Type Culture Collection

[0056] Abbreviation of the depositary institution: CCTCC

[0057] Address: Wuhan University, Wuhan, China

[0058] Date of deposit: October 30, 2024

[0059] Taxonomic name: Muscovy duck reovirus ( Muscovy Duck Reovirus )

[0060] Registration number in the depositary center: CCTCC NO: V202491.

[0061] Example 2: Investigation on the characteristics of goose-origin Muscovy duck reovirus JS001

[0062] 1. Pathogenicity of the virus:

[0063] Sixty 5-day-old healthy goslings (Zhedong white geese) were randomly divided into two groups: the experimental group and the control group. The experimental group was intramuscularly injected with the virus solution of goose-origin Muscovy duck reovirus JS001, 0.5 mL (TCID 50 was 10 -6.2 / 0.1 mL) / goose; the control group was injected with an equal amount of sterile normal saline, and the clinical manifestations of each group of goslings were observed for 14 days.

[0064] On the 2nd day after virus challenge, the goslings in the experimental group began to get sick, with an incidence rate of 100% and a mortality rate reaching 50%. The infected geese were listless, anorectic, had movement disorders, excreted yellowish-green loose stools, and showed symptoms of swollen joints. Autopsy showed a large number of white necrotic spots on the liver and spleen and severe hemorrhage in the joint cavity.

[0065] All the goslings in the control group were healthy and alive, without any clinical manifestations. After autopsy 14 days later, no lesions were found.

[0066] It can be seen that the goose-origin Muscovy duck reovirus JS001 of the present invention has the ability to cause "flower liver disease" and joint cavity hemorrhage in goslings.

[0067] 2. Immunogenicity of the virus:

[0068] After diluting the goose-origin Muscovy duck reovirus JS001 by 5 times, attenuated live virus antigen and inactivated antigen were prepared, and healthy 5-day-old goslings without vaccination were immunized respectively. The sera were collected at 5 d, 10 d, and 20 d after immunization, and at 7 d and 12 d after virus challenge. The antibodies in the sera were detected by indirect ELISA method.

[0069] The results showed that the antibody level of goslings immunized with live virus antigen was lower than that of goslings immunized with inactivated antigen; that is, the inactivated antigen had better immunogenicity than the live virus antigen.

[0070] The inactivated vaccine prepared from the goose-origin Muscovy duck reovirus JS001 was inoculated into adult female breeding geese. The offspring produced by the geese immunized with inactivated antigen could all generate complete protection against virus challenge, indicating that the JS001 strain of the goose-origin Muscovy duck reovirus had excellent immunogenicity.

[0071] 3. Cell tropism

[0072] To determine the cell tropism of the goose-origin Muscovy duck reovirus JS001, the method of observing cytopathic effect was used in this study to detect the proliferation of the goose-origin Muscovy duck reovirus JS001 on LMH cells and Vero cells, and the virus growth curve of the JS001 strain was made in LMH cells ( Figure 3 ), as follows:

[0073] (1) Test method:

[0074] The goose-origin Muscovy duck reovirus JS001 strain was inoculated onto LMH cells with good growth state, adsorbed in the cell incubator for 30 min, the sample treatment solution was discarded, and 2% cell maintenance solution was added for continued culture; and a negative control was set with sterile physiological saline and incubated at 37 °C. The cell state was observed and recorded daily. When the cell fusion reached 80%, the virus solution was collected, and the virus content was calculated by the Reed-Muench method.

[0075] (2) The results of the virus content of the goose-origin Muscovy duck reovirus JS001 strain proliferated on different cells are shown in Table 2.

[0076] Table 2: The virus content of the goose-origin Muscovy duck reovirus JS001 strain proliferated on different cells

[0077]

[0078] The purification and culture of Muscovy duck reovirus from geese is a difficult point in virus and vaccine research. Whether a vaccine strain adapted to in vitro cell culture can be obtained is the key to vaccine development and production. The Muscovy duck reovirus JS001 from geese in the present invention has a wide cell tropism, can infect a variety of animal cells, and can produce a large number of proliferations in a short time. Therefore, the Muscovy duck reovirus JS001 strain in the present invention can be used as an excellent vaccine strain for the preparation of vaccines.

[0079] Example 3: Preparation of inactivated vaccine

[0080] (1)Virus proliferation and harvest:

[0081] The Muscovy duck reovirus JS001 after isolation and identification was inoculated into well-grown LMH cells, and the inoculation volume ratio was 1:100; adsorbed at 37°C for 30 min, and then cultured with DMEM containing 2% newborn bovine serum under the conditions of 37°C and 5% CO2 until 80% of the cells showed cytopathic effects and then harvested to obtain cell virus fluid. The obtained sufficient cell virus fluid was frozen at -20°C, and after two freeze-thaw cycles, the supernatant was collected by centrifugation to obtain virus fluid. Calculate TCID 50 , the virus content in each 0.1 ml of virus fluid is 10 -6.2 TCID 50 .

[0082] (2)Virus purification:

[0083] The established detection methods such as PCR and RT-PCR were used to detect whether other common viruses were contained in the obtained virus fluid. The detection items included DRV, TMUV, DAStV, DuCV, DPV, and DHAV, etc., and the seed virus was purified.

[0084] (3)Inactivation of virus fluid:

[0085] The virus fluid with qualified bacterial inspection was inactivated with formaldehyde, and the best inactivation condition was to add formaldehyde with a final concentration of 0.2% and inactivate it by stirring at 37°C for 16 h.

[0086] (4)Vaccine preparation:

[0087] ① Preparation of oil phase: Take medicinal white oil No. 10 and Span-80 in a ratio of 94:6 and mix them evenly, then add 2% aluminum tristearate, stir until light yellow and clear and transparent, and then sterilize it by high-pressure steam at 121°C for standby.

[0088] ② Preparation of the aqueous phase: The completely inactivated virus solution and sterile Tween-80 are mixed by shaking in a ratio of 96:4 to completely dissolve Tween-80.

[0089] ③ Emulsification: The oil phase and the aqueous phase are mixed in a ratio of 2:1. Add 2 parts of the oil phase to the tissue homogenizer in a laminar flow hood, and slowly add 1 part of the aqueous phase while continuously stirring. After all the aqueous phase is added, mix at 6000 rpm for 10 min, and then emulsify at 8000 r / min for 20 min. Dispense and store for later use, that is, the inactivated vaccine is prepared.

[0090] Example 4: Quality inspection of the inactivated vaccine

[0091] Perform quality inspections on the inactivated vaccine prepared in Example 3, including: dosage form, centrifugal stability, viscosity, sterility, and shelf life. The specific methods refer to the "Pharmacopoeia of the People's Republic of China" (2015 Edition).

[0092] The results are as follows: The dosage form of the inactivated vaccine prepared in the present invention is water-in-oil (W / O); the centrifugal stability, viscosity, and sterility inspections meet the regulations of the "Pharmacopoeia of the People's Republic of China" (2015 Edition).

[0093] Example 5: Safety inspection of the inactivated vaccine

[0094] Take 20 5-day-old goslings, randomly divide them into 2 groups evenly, with 10 in each group. Among them, the first group is the experimental group, and the gosling-origin Muscovy duck reovirus prepared in Example 3 is injected into the leg muscle at 0.5 mL per bird. The second group is the control group, and an equal amount of sterilized white oil adjuvant is injected into the leg muscle. After inoculation, observe the mental state of each group of animals every day, and whether there are local inflammatory reactions such as redness, swelling, heat, and pain at the injection site. Continuously observe for 2 weeks. After 2 weeks, dissect the experimental animals to observe the absorption of the vaccine at the injection site;

[0095] Results: The experimental group showed a brief listlessness, but quickly recovered. After continuous observation for two weeks, the growth and development of both the experimental group and the control group were normal, with good mental states. Autopsy of the experimental group found that the vaccine was well absorbed at the injection site, without inflammatory reactions such as redness, swelling, and tissue necrosis. The results prove that the trial-produced vaccine is safe and harmless and has no impact on animal growth.

[0096] Example 6: Protective inspection of the inactivated vaccine

[0097] Take 40 5-day-old goslings, randomly divide them into 2 groups evenly, with 20 in each group. Among them, the first group is the immunized group, and the inactivated vaccine prepared in Example 3 is injected into the leg muscle at 0.5 mL per bird; the second group is the control group, and an equal amount of sterilized white oil adjuvant is injected into the leg muscle. 10 days after immunization, inject 0.2 mL of gosling-origin Muscovy duck reovirus solution into the legs of the two groups of goslings, and raise them separately in groups, and observe the mental status and death of the two groups of goslings.

[0098] Results: The immunized group showed a transient listlessness, but did not show necropsy changes of "flower liver disease" (white necrotic spots appeared on the surfaces of the liver and spleen) and bleeding in the joint cavity, and no gosling died; the goslings in the control group showed obvious clinical symptoms of swollen joint cavities after challenge. Necropsy showed that a large number of white necrotic spots appeared on the surfaces of the liver and spleen and there was bleeding in the joint cavity, and the number of dead goslings was 8. The results showed that the immune protection rate of the inactivated vaccine prepared by the present invention could reach 100%.

[0099] Example 7: Determination of the immune persistence period of the inactivated vaccine

[0100] Forty 5-day-old goslings were randomly divided into 2 groups, with 20 goslings in each group. Among them, the first group was the immunized group, and each gosling was injected with the inactivated vaccine prepared in Example 3 into the leg muscle at a dose of 0.5 mL / gosling; the second group was the control group, and the leg muscle was injected with an equal amount of sterilized white oil adjuvant; sera were collected every two days after immunization and detected for antibodies by indirect ELISA.

[0101] It was found that the antibody level in the immunized group began to increase significantly on the 4th day, reached the peak on the 8th day, and then decreased slightly but still remained at a relatively high level within 45 days; while the antibody level in the control group was negative throughout the experiment. This indicates that the inactivated vaccine of the present invention can rapidly reach a high concentration of antibody level in a short time, and has a long immune persistence period, and can provide rapid and long-term immune protection for goslings.

[0102] The above are only the preferred embodiments of the present application and are not intended to limit the present application. For those skilled in the art, various modifications

[0103] equivalent replacements, improvements, etc. should be included in the protection scope of the present application.

Claims

1. An inactivated vaccine for preventing and treating goose-origin Muscovy duck reovirus, characterized in that, The inactivated vaccine contains an inactivated Muscovy duck reovirus solution of goose origin with a virus titer of 10 -6.2 TCID 50 / 0.1 mL; the Muscovy duck reovirus of goose origin was deposited at the China Center for Type Culture Collection on October 30, 2024, with the deposit number: CCTCC NO: V202491; The inactivated vaccine is used for preventing and treating "flower liver disease" and joint cavity bleeding in goslings; The preparation method of the inactivated goose-origin Muscovy duck reovirus liquid comprises the following steps: (1) Inoculate the goose-origin Muscovy duck reovirus strain with the preservation number of CCTCC NO: V202491 into LMH cells. After proliferating and culturing the goose-origin Muscovy duck reovirus, break the cells by freeze-thawing, collect the supernatant, and purify it to obtain the goose-origin Muscovy duck reovirus liquid; (2) Add formaldehyde with a final concentration of 0.2% to the goose-origin Muscovy duck reovirus liquid obtained in step (1), and inactivate it by stirring at 37°C for 16 h to obtain the inactivated goose-origin Muscovy duck reovirus liquid; The preparation method of the inactivated vaccine comprises the following steps: Take the inactivated goose-origin Muscovy duck reovirus liquid, add Tween-80 and mix it as the water phase, take white oil, aluminum stearate and Span-80 and mix them as the oil phase, mix the oil phase and the water phase evenly at a ratio of 2:1, and emulsify to obtain the inactivated vaccine for preventing and treating goose-origin Muscovy duck reovirus.

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