Duck Tembusu virus CHN-YC, its bivalent inactivated vaccine and preparation method

Abstract

The present invention discloses a duck Tembusu virus CHN-YC and a dual inactivated vaccine thereof and a preparation method. The deposit number of CHN-YC is CCTCC NO: V202371. The vaccine comprises an inactivated duck Tembusu virus CHN-YC and an inactivated novel duck reovirus QR-China / 2020. The present invention obtains a highly pathogenic duck Tembusu virus strain, and the inoculation dose of the virus to 5-day-old SPF ducks is 10 7 TCID 50 The lethality rate reaches 55%. Furthermore, the strain has strong immunogenicity and is suitable for use as a vaccine strain. The dual inactivated vaccine of the present invention is relatively stable, enabling ducks injected with the vaccine to produce high levels of serum neutralizing antibodies, thereby achieving better immunization against the novel duck reovirus and duck Tembusu virus. Consequently, ducks injected with the dual inactivated vaccine are less susceptible to diseases caused by the two viruses, allowing for simultaneous immunization against two diseases.

Description

Technical Field

[0001] The present invention relates to the field of veterinary inactivated vaccines, and in particular to a duck Tembusu virus CHN-YC and a combined inactivated vaccine thereof and a preparation method thereof. Background Art

[0002] Duck Tembusu virus (DTMUV) is a recently emerging avian virus, a member of the Flaviviridae family and genus Flavivirus. Since its discovery in duck farms in Guangdong Province, China in 2010, DTMUV has spread rapidly throughout China, causing severe losses to the poultry industry. The disease primarily causes depression in laying ducks and a sharp drop in egg production; broiler ducks experience weight loss or even weight loss, and a loss of appetite; and ducklings develop high fever, growth retardation, lethargy, and a high mortality rate.

[0003] Novel duck reovirus (NDRV) is a virus that has emerged in recent years and has had a serious impact on waterfowl. This virus belongs to the Reoviridae family and the Orthoreovirus genus and can cause duck hemorrhagic necrotizing hepatitis. The virus appeared in my country in 2005, became prevalent in southern my country, and continues to spread widely, causing serious losses to my country's duck farming industry. The pathogen is clearly different from avian orthoreovirus (ARV) and Muscovy duck reovirus (MDRV), and is a new type of duck reovirus. Infected ducks are characterized by hemorrhagic necrosis of the liver, and symptoms such as slow growth, depression, decreased appetite, clustering and squatting, and white loose feces.

[0004] There is currently no dual inactivated vaccine for duck Tembusu virus and new duck reovirus on the market in my country. Therefore, the development of an efficient dual inactivated vaccine meets the current market demand of the duck farming industry and is of great significance for the prevention and control of duck Tembusu virus and new duck reovirus in my country. Summary of the Invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a duck Tembusu virus CHN-YC and a bivalent inactivated vaccine thereof and a preparation method. The bivalent inactivated vaccine has the advantages of simultaneously immunizing against novel duck reovirus disease and duck Tembusu virus disease.

[0006] To achieve the above purpose, the technical solution designed by the present invention is as follows:

[0007] The present invention provides a duck Tembusu virus CHN-YC, whose deposit number is CCTCC NO: V202371.

[0008] The duck Tembusu virus (CHN-YC) was deposited with the China Center for Type Culture Collection (CCTCC) at Wuhan University in Wuhan, Hubei Province, on June 24, 2023, under the deposit number CCTCC NO: V202371. The duck Tembusu virus strain can be used to prepare vaccines or antibodies for preventing duck Tembusu virus-induced significant suppression of weight gain in ducks, decreased egg production in ducks, neurological symptoms in ducklings, and even death.

[0009] The present invention also provides a use of the duck Tembusu virus CHN-YC in preparing a vaccine for preventing and treating duck Tembusu virus.

[0010] The present invention also provides a dual inactivated vaccine of duck Tembusu virus and novel duck reovirus, wherein the dual inactivated vaccine comprises inactivated duck Tembusu virus CHN-YC and inactivated novel duck reovirus (Novel DuckReovirus) QR-China / 2020; wherein, the preservation number of the duck Tembusu virus CHN-YC is: CCTCC NO: V202371; the preservation number of the novel duck reovirus QR-China / 2020 is: CCTCC NO: V202372.

[0011] The above-mentioned novel duck reovirus (Novel Duck Reovirus) QR-China / 2020 was deposited in the China Center for Type Culture Collection on June 24, 2023. The deposit address is Wuhan University, Wuhan City, Hubei Province, and the deposit number is: CCTCC NO: V202372.

[0012] Furthermore, in the said two-inactivated vaccine, the content of duck Tembusu virus CHN-YC is 10 7 ~10 8 TCID 50 / mL, the content of novel duck reovirus QR-China / 2020 was 10 7 ~10 8 TCID 50 / mL, and the volume ratio of duck Tembusu virus CHN-YC to new duck reovirus QR-China / 2020 was 1:0.5~2.

[0013] Furthermore, in the said two-inactivated vaccine, the content of duck Tembusu virus CHN-YC is 10 7.7 TCID 50 / mL, the content of novel duck reovirus QR-China / 2020 was 10 7.1 TCID 50 / mL, and the volume ratio of duck Tembusu virus CHN-YC to the new duck reovirus QR-China / 2020 strain was 1:1.

[0014] Furthermore, the inactivated vaccine also includes a pharmaceutically acceptable carrier.

[0015] Further, the carrier is an adjuvant;

[0016] Furthermore, the adjuvant is any one or more of white oil, Span-80, Tween-80 and aluminum stearate.

[0017] The present invention also provides a method for preparing the above-mentioned duck Tembusu virus and novel duck reovirus combined inactivated vaccine, comprising the following steps:

[0018] 1) Preparation of Duck Tembusu Virus CHN-YC Fluid

[0019] The duck Tembusu virus CHN-YC strain was inoculated on BHK-21 cells at a dose of 0.01 to 0.1 MOI by adsorption inoculation, and the virus was collected 48 to 60 hours after inoculation. The virus titer of the virus solution was determined to be 10 -6.7 / 0.1mL;

[0020] 2) Preparation of the novel duck reovirus QR-China / 2020 virus solution

[0021] The novel duck reovirus QR-China / 2020 strain was inoculated on Vero cells at a dose of 0.1-0.2 MOI using a non-adsorption inoculation method, and the virus was collected 72-96 hours after inoculation. After the virus was collected, the virus titer of the virus liquid was measured, and the virus titer of the novel duck reovirus QR-China / 2020 strain was measured to be 10 -6.1 / 0.1mL;

[0022] 3) Preparation of mixed virus solution

[0023] The virus solution of duck Tembusu virus CHN-YC and the virus solution of novel duck reovirus QR-China / 2020 were mixed at a volume ratio of 1:0.5-2; the inactivated mixed virus solution was obtained by inactivation.

[0024] 4) Preparation of the combined inactivated vaccine

[0025] a. Weigh 94 parts of medical white oil, 6 parts of Siben-80 and 2 parts of aluminum stearate in parts by weight, mix well and sterilize at 121 ° C and autoclave for 15 minutes, cool to room temperature to obtain an oil phase and set aside;

[0026] b. Weigh 96 parts by weight of the inactivated mixed virus solution and 4 parts of sterilized Tween-80; stir evenly as the aqueous phase;

[0027] c. Weigh the oil phase and the water phase in a volume ratio of 2:1; slowly stir the oil phase in a high-speed emulsifier while slowly adding the water phase. After the addition, stir at a speed of 10,000 r / min for 5 to 15 minutes to obtain a two-inactivated vaccine. After packaging, store it at 4°C. The content of duck Tembusu virus CHN-YC in the vaccine is 10 7 ~10 8 TCID 50 / mL, the content of novel duck reovirus QR-China / 2020 was 10 7 ~10 8 TCID 50 / mL.

[0028] Beneficial effects of the present invention:

[0029] 1. The present invention has isolated a strain of duck Tembusu virus with strong pathogenicity, and the dose of the virus was 10 7 TCID 50 The lethality rate reaches 55%. The strain has strong immunogenicity and is suitable for use as a vaccine strain.

[0030] 2. The bivalent inactivated vaccine prepared by the present invention has relatively stable properties, so that ducks injected with the vaccine can produce high levels of serum neutralizing antibodies, thereby achieving better immunization effects against the novel duck reovirus and duck Tembusu virus, thereby making the ducks injected with the bivalent inactivated vaccine less susceptible to diseases caused by the novel duck reovirus and diseases caused by duck Tembusu virus. The vaccine can be used to immunize against two diseases at the same time. When a disease breaks out, the use of the vaccine reduces the cost of the duck farming industry, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the identification diagram of the isolated new duck reovirus;

[0032] In the figure, MOCK represents cells not infected with the virus, and QR represents cells infected with the novel duck reovirus QR-China;

[0033] Figure 2 This is the identification diagram of the isolated duck Tembusu virus;

[0034] In the figure, MOCK represents cells not infected with the virus, and CHN-YC represents cells infected with duck Tembusu virus CHN-YC;

[0035] Figure 3The cytopathic effect of BHK-21 cells infected with CHN-YC is shown in Figure 2.

[0036] Figure 4 This is the cytopathic effect image of Vero cells infected with QR;

[0037] Figure 5 This is the growth curve of CHN-YC on different cells;

[0038] Figure 6 Growth curves of CHN-YC (A) and QR (B) inoculated with different doses;

[0039] Figure 7 This is a picture of the clinical manifestations of ducklings infected with DTMUV;

[0040] In the picture, A: The duckling has its eyes closed and cannot sit or lie down; B: The duckling has its head and neck tilted; C: The duckling has green, liquid feces;

[0041] Figure 8 Figure 2 shows the weight change (A) and survival curve (B) of ducklings in the observation group;

[0042] Figure 9 This is the detoxification pattern of ducklings in the infected group;

[0043] Figure 10 The following are the lesion images of the ducks in the infection group;

[0044] In the figure, A: spleen of infected ducklings; B: brain of infected ducklings; C: heart of infected ducklings; D: liver of infected ducklings;

[0045] Figure 11 This is the histopathological observation of the duck organs in the infection group

[0046] In the figure, A: pathological section of spleen of infected ducklings; B: pathological section of liver of infected ducklings; C: pathological section of brain of infected ducklings;

[0047] In the figure, A: pathological section of spleen of ducklings in the infection group;

[0048] B: Pathological section of liver of ducklings in the infection group;

[0049] C: Pathological sections of brain in the infected group;

[0050] Figure 12 This is a diagram of the virus distribution in different tissues after DTMUV infection;

[0051] In the figure, A is the DTMUV content in the serum of ducks in different strain groups;

[0052] B is the graph of DTMUV content in duck spleens of different strain groups;

[0053] C is the graph of DTMUV content in duck brains of different strain groups;

[0054] D is the graph of DTMUV content in duck hearts of different strain groups;

[0055] E is the graph of DTMUV content in duck kidneys of different strain groups;

[0056] F is the graph of DTMUV content in duck livers of different strain groups.

[0057] Figure 13 This is the antibody detection graph in the serum of the infected group;

[0058] Figure 14 Figure 2 is the result of exogenous virus detection in CHN-Y (A) and QR (B);

[0059] In the figure, M: DNA Marker; 1(A): DTMUV; 1(B): NDRV; 2: MDV; 3: DPMV; 4: MDRV; 5: DHAV; 6: EDSV; 7: DAdV-3; 8: NDV;

[0060] Figure 15 This is an autopsy picture of a single-dose vaccine safety trial;

[0061] In the figure, A: control group; B: single-dose group; C: double-dose group;

[0062] Figure 16 Autopsy images for repeated vaccine immunization safety trials;

[0063] A: control group; B: single-dose group; C: double-dose group;

[0064] Figure 17 This is a graph showing the changes in antibody levels in immunized duck serum;

[0065] In the figure, A: blocking ELISA to detect anti-DTMUV antibodies in serum; B: serum neutralization test to detect anti-DTMUV neutralizing antibodies in serum; C: blocking ELISA to detect anti-NDRV antibodies in serum; D: serum neutralization test to detect anti-NDRV neutralizing antibodies in serum;

[0066] Figure 18 The viral load detection graphs of DTMUV (A) and NDRV (B) in serum after challenge;

[0067] Figure 19 Figure 2 is a graph showing the daily weight gain changes after DTMUV (A) and NDRV (B) challenge;

[0068] Figure 20 The graph shows the changes of anti-DTMUV antibodies (A) and neutralizing antibodies (B) in the serum of the single immunization group;

[0069] Figure 21 The graph shows the changes of anti-DTMUV antibodies (A) and neutralizing antibodies (B) in the serum of the repeated immunization group;

[0070] Figure 22 The graph shows the changes of anti-NDRV antibodies (A) and neutralizing antibodies (B) in the serum of the single immunization group;

[0071] Figure 23 Graph showing changes in anti-NDRV antibodies (A) and neutralizing antibodies (B) in the serum of the repeated immunization group. DETAILED DESCRIPTION

[0072] The present invention is further described in detail below with reference to specific embodiments so that those skilled in the art can understand.

[0073] Example 1 Isolation and Identification of Duck Tembusu Virus CHN-YC and Novel Duck Reovirus QR-China / 2020

[0074] Diseased tissues were collected from a duck farm in Hubei Province. Three to five volumes of DMEM medium and 100 μg / mL of penicillin and 100 μg / mL of streptomycin were added at 4°C. The tissues were ground with a homogenizer and then frozen and thawed three times at -80°C. The tissues were then centrifuged at 12,000 rpm for 10 minutes at 4°C. The supernatants were collected and tested using IFA. The two strains of the virus were found to be duck Tembusu virus and a novel duck reovirus. RNA from the plaque-purified duck Tembusu virus and novel duck reovirus was then extracted and reverse-transcribed into cDNA. Primers were designed to sequence the entire viral genome, and homology analysis was performed with other strains.

[0075] At the same time, if Figures 1-2 As shown, when BHK-21 cells were not inoculated with the virus, no specific green fluorescence was observed when the cells were observed under a microscope using an indirect immunofluorescence assay. However, when the BHK-21 cells were inoculated with the virus, specific green fluorescence was observed through reaction with anti-Tembusu virus antibodies, indicating that the virus was duck Tembusu virus. When Vero cells were not inoculated with the virus, no green fluorescence was observed. However, when another virus isolated from diseased duck farm material was inoculated onto the cells, specific green fluorescence was observed under a microscope using an indirect immunofluorescence assay, indicating that the virus was a novel duck reovirus.

[0076] The duck Tembusu virus CHN-YC (hereinafter referred to as CHN-YC) and the novel duck reovirus QR-China / 2020 (hereinafter referred to as QR) were identified;

[0077] The duck Tembusu virus (CHN-YC) was deposited with the China Center for Type Culture Collection (CCTCC) at Wuhan University in Wuhan, Hubei Province, on June 24, 2023, under the deposit number CCTCC NO: V202371. The duck Tembusu virus strain can be used to prepare vaccines or antibodies for preventing duck Tembusu virus-induced significant suppression of weight gain in ducks, decreased egg production in ducks, neurological symptoms in ducklings, and even death.

[0078] The above-mentioned new duck reovirus (Duck Reovirus) QR-China / 2020 was deposited in the China Center for Type Culture Collection on June 24, 2023. The collection address is Wuhan University, Wuhan City, Hubei Province, and the collection number is: CCTCC NO: V202372.

[0079] Example 2

[0080] 1. Recovery, Passaging, and Cryopreservation of Inoculated Cells

[0081] 1.1 Recovery of BHK-21 and Vero cells

[0082] (1) Preheat 10% FPB DMEM / MEM medium to 37°C in a water bath.

[0083] (2) Take out the desired frozen cells from the liquid nitrogen tank and quickly place them in a 30°C water bath until the frozen cell liquid melts.

[0084] (3) Add the thawed cell solution into 5 mL of preheated 10% FPB DMEM / MEM medium and centrifuge at 1000 rpm for 5 minutes.

[0085] (4) Discard the supernatant, add 5 mL of 10% FPB DMEM / MEM medium, and gently resuspend until the cell clumps are completely dispersed.

[0086] (5) Place the resuspended cells into a T25 cell culture flask, mark it, mix well, and place it flat in a 37°C, 5% CO2 incubator for culture.

[0087] (6) After 12 hours, decide whether to change the medium based on the cell status.

[0088] 1.2 Cell passaging

[0089] (1) Preparation: Preheat PBS, trypsin, and 10% FPB DMEM / MEM medium in a 37°C water bath.

[0090] (2) Washing: Take out the cells that have grown to 95% or more from the incubator, discard the culture medium in the cell bottle in the clean bench, and add an appropriate amount of sterile PBS to wash the cells twice.

[0091] (3) Digestion: Add an appropriate amount of trypsin to the cell flask, shake the cell flask horizontally so that the trypsin can cover all the cells, discard the trypsin after shaking several times, and place the cell flask upside down and flat in a 37°C incubator. Generally, after 5 minutes (different cells have different digestion times, depending on the specific cells), the cells move in a sand-like manner when observed with the naked eye. Under a microscope, if the intercellular gap increases and the cells become round, it means that the cells have been completely digested.

[0092] (4) Dispersion: Add 5 mL of 10% FPB DMEM / MEM medium to the cell flask to neutralize the trypsin, and use an electric pipette to gently blow the cells on the wall of the cell flask until the cells are dispersed into single cells or uniform groups of three or five.

[0093] (5) Passaging: Take an appropriate amount of cell suspension as needed, add culture medium, shake well, and place flat in a 37°C, 5% CO2 incubator for culture.

[0094] 1.3 Cell cryopreservation

[0095] (1) Preparation of freezing solution: 10% DMSO + 90% FBS, mix well and equilibrate in a 37°C water bath.

[0096] (2) Cryopreservation: After the cells are dispersed according to the cell passage procedure, the cell suspension used is collected into a 15 mL centrifuge tube, centrifuged at 1000 rpm for 5 min at room temperature, and the supernatant is discarded. An appropriate amount of freezing solution is added and the suspension is gently resuspended. The cell suspension is evenly distributed into cryopreservation tubes, with an average of 1 mL per tube. After labeling, the tubes are placed in a cryopreservation box. First, the tubes are placed in a -20 °C refrigerator for 2 h, then in a -80 °C refrigerator for 24 h, and then transferred to a liquid nitrogen tank for long-term storage.

[0097] 2. Virus Passaging

[0098] 2.1 Virus inoculation and observation

[0099] The T25 cell flask filled with a monolayer of BHK-21 cells and Vero cells was taken out and placed in a clean bench. The supernatant was discarded and the cells were washed twice with sterile PBS. Then, the virus solution containing an appropriate amount of virus was added to the cell flask and the total volume was made up to 2.5 mL with cell maintenance solution. The cells were placed in a 37°C, 5% CO2 incubator. After 2 hours, the virus solution was discarded and 5 mL of cell maintenance solution was added. The cells were cultured in a 37°C, 5% CO2 incubator. After 36 to 48 hours, the cell flask was placed under a microscope to observe the virus-induced cytopathic effect.

[0100] 3. Results:

[0101] When CHN-YC was inoculated on BHK-21 cells grown to a monolayer, after 48 hours, it was observed that the cell transparency decreased, the cells became rounded and shrunken, and aggregated in grape-like clusters, and the cytoplasm was vacuolated. Figure 3 When QR was inoculated on Vero cells that had grown to a monolayer for 48 hours, it was observed that the cells shrank and aggregated, fell off in large numbers, lost their normal cell morphology, and formed a network structure, as shown in Figure 2. Figure 4 shown.

[0102] 4. Virus Collection and Storage

[0103] When the cytopathic effect reaches 90% or above, place the cell bottle inoculated with the virus (CHN-YC / QR) at -80°C and freeze-thaw three times repeatedly. Then, divide the virus liquid in the cell bottle into 1.5 mL centrifuge tubes, centrifuge at 12000 r / min, 4°C for 10 minutes, and take the supernatant after centrifugation into 1.5 mL sterile centrifuge tubes. After labeling, store them in an 80°C refrigerator.

[0104] Example 3 CHN-YC Optimal Seeding Cells

[0105] 1. Virus plaque assay

[0106] (1) Cells were seeded into 6-well plates and placed in a 37°C, 5% CO2 incubator until the cells grew into a confluent monolayer.

[0107] (2) The virus sample to be tested was diluted 10-fold in a sterile 1.5 mL EP tube.

[0108] (3) Aspirate the cell culture supernatant in the 6-well plate, wash three times with PBS, add the virus solution diluted with virus growth medium (generally three dilutions are tested for each sample to be tested), mix gently, and incubate in a 37°C, 5% CO2 incubator for 2 hours.

[0109] (4) Preparation of plaque solution: Mix phenol red-free 2×MEM and 1.8% low-melting point agarose in a ratio of 1:1, add 1% double antibody and 2% FBS, mix well and set aside.

[0110] (5) Aspirate the virus solution in the 6-well plate and wash away the unadsorbed virus particles and dead cells with PBS buffer containing calcium and magnesium.

[0111] (6) Drain the liquid in the 6-well plate and add the prepared plaque solution, taking care not to introduce air bubbles. Place the 6-well plate in a 4°C refrigerator to condense (10-15 minutes) and then place it in a 37°C, 5% CO2 cell culture incubator for 2-3 days.

[0112] (7) After the spots appear, use a sterile pipette tip to pick out a single virus plaque, then expand and culture the picked virus plaque on cells, observe its pathological changes, and identify it using RT-PCR.

[0113] (8) Staining: After plaque formation, fix with 4% paraformaldehyde at 37°C for 15 minutes, add 2 mL of PBS, and pick out the agarose covering layer; add 1% crystal violet to completely cover the cells and stain for 1 hour; finally, rinse with PBS to remove the crystal violet, dry, and observe the morphology of viral plaques on a whiteboard light and take pictures.

[0114] 2. TCID 50 )

[0115] The virus solution to be tested was diluted 10-fold in a 1.5 mL EP tube with the maintenance solution, i.e., 10 -1 ~10 -10 Etc., the supernatant of animal serum and tissue homogenate was filtered through a 0.22μm sterilizing filter and diluted 10 times. The virus solution of each dilution was inoculated into columns 1 to 10 in a 96-well plate, with 8 wells in a column and 100μL inoculated into each well. An equal volume of maintenance solution was added to columns 11 to 12 as a control. The cells that grew a single layer were digested and diluted with maintenance solution and added to the above-mentioned 96-well plate with a spray gun at a ratio of 1:2. The 96-well plate was placed in a 37°C, 5% CO2 incubator for culture. Observation began after 72 hours, and the number of CPE holes produced by the cells was recorded until no CPE holes appeared after about 96 hours. The above experiment was repeated three times, and the TCID of the virus to be tested was calculated according to the Reed-Muench method. 50 value.

[0116] 3. Screening of the best seeding cells for CHN-YC

[0117] When BHK-21 cells and DF-1 cells grew to about 80%, the same dose of CHN-YC was inoculated on these two cells and placed in a 37°C, 5% CO2 incubator. After 2 hours, the virus solution was discarded and cell maintenance medium was added. The cells were cultured in a 37°C, 5% CO2 incubator. The virus was collected at 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, and 96 hours, and the TCID 50 The determination was performed by comparing the viral TCID 50 The optimal seeding cells of CHN-YC were screened out by adjusting the level of

[0118] Results: The TCID of CHN-YC on BHK-21 cells and DF-1 cells at different time points were measured. 50 The growth curves of CHN-YC on different cells were determined. Figure 5 As shown. Figure 5 It was found that after the virus was inoculated into BHK-21 cells, the virus titer continued to rise within 60 hours, and the highest virus titer at 60 hours was 10 7.4 TCID 50 / mL, and the virus titer began to decline after 60h. After CHN-YC was inoculated into DF-1 cells, the highest point of virus titer was 72h, reaching 10 6.4 TCID 50 / mL, and the virus titer began to decline after 72h. Figure 5 It can be seen that when BHK-21 cells were used as the inoculated cells for CHN-YC, the highest viral titer was much higher than that when inoculated on DF-1 cells, and the time when the highest viral titer was reached was earlier than that when inoculated on DF-1 cells. BHK-21 cells are easier to culture than DF-1 cells. In summary, BHK-21 cells were determined to be the best inoculated cells for CHN-YC.

[0119] Example 4 Optimization of CHN-YC and QR Proliferation Conditions

[0120] (1) BHK-21 cells and Vero cells were inoculated into cell culture flasks using MEM culture medium and DMEM culture medium containing 10% FBS, respectively, and cultured in a 37°C, 5% CO2 incubator until the cells grew into a monolayer, after which the growth medium was discarded.

[0121] (2) Melt the CHN-YC strain and the QR strain on ice, inoculate them onto BHK-21 cells and Vero cells at the following concentrations (MOI = 0.1, MOI = 0.01, MOI = 0.001), respectively, and incubate them in a 37°C incubator for 2 h. Every 30 min, remove the cells from the incubator, shake them slightly, and then put them back into the incubator to allow complete virus adsorption.

[0122] (3) Discard the inoculum, wash with sterile PBS, add MEM medium containing 2% FBS to BHK-21 cells, add DMEM medium containing 2% FBS to Vero cells, and then place in a 37°C, 5% CO2 incubator for culture.

[0123] (4) Collect the poison at 12 h, 24 h, 36 h, 48 h, 60 h, 72 h, 84 h, and 96 h, respectively, divide the contents into 1.5 mL EP tubes, label them, and store them in a -80 °C refrigerator for later use.

[0124] (5) The virus titers of CHN-YC and QR propagated on different cells at different time periods were measured respectively, and growth curves were drawn to determine the optimal virus inoculation dose and the optimal virus collection time for mass virus propagation.

[0125] Results: CHN-YC and QR were inoculated on BHK-21 cells and Vero cells at the following concentrations (MOI = 0.1, MOI = 0.01, MOI = 0.001), and the viruses were collected at different time points and TCID 50 The growth curves of CHN-YC and QR inoculated with different virus doses were plotted. Figure 6 The inoculation dose of CHN-YC was 0.1 MOI. When the virus was collected for 36 hours, the virus titer reached the highest level, reaching 10 7.0 TCID 50 / mL, and then the virus titer continued to decline; when the inoculation dose was 0.01 MOI and the virus collection time was 60h, the highest virus titer could reach 10 7.6 TCID 50 / mL; the inoculation dose is 0.001MOI, and the virus titer can reach up to 10 when the virus is collected for 72h. 6.9 TCID 50 / mL, so the optimal inoculation dose of CHN-YC is 0.01MOI, and the optimal collection time is 60h after inoculation. When the inoculation dose of QR is 0.1MOI and the collection time is 72h, the virus titer is the highest, reaching 10 5.9 TCID 50 / mL; the inoculation dose is 0.01MOI, and the virus titer can reach up to 10 when the virus is collected for 84h. 4.9 TCID 50 / mL; the inoculation dose is 0.001MOI, and the virus titer can reach up to 10 when the virus collection time is 96h. 4.3 TCID 50 / mL, so the optimal dose of QR is 0.1MOI, and the optimal time of collection is 72h after infection.

[0126] Example 5 Screening of the Optimal Inoculation Methods for CHN-YC and QR

[0127] BHK-21 cells and Vero cells were seeded into cell culture flasks using MEM medium and DMEM medium containing 10% FBS, respectively. The cells were cultured in a 37°C, 5% CO2 incubator until the cells reached a monolayer, after which the growth medium was discarded. The CHN-YC and QR strains were then thawed on ice and inoculated using the following three methods:

[0128] (1) Synchronous inoculation method: Wash the grown cells twice with PBS and then digest them with trypsin. Add the virus solution to the cell maintenance medium in a certain proportion. Then add the virus-added maintenance medium to the digested cells and culture them in a 37°C, 5% CO2 incubator.

[0129] (2) Adsorption inoculation method: Add the virus solution to the cell maintenance medium in a certain proportion, add half the volume of the cell maintenance medium to the cells that have grown into a monolayer, place it in a 37°C incubator for 2 hours, discard the liquid, add the normal volume of cell maintenance medium, and then place it in a 37°C, 5% CO2 incubator for culture.

[0130] (3) Non-adsorption inoculation method: Add the virus solution to the cell maintenance medium in a certain proportion, add the cell maintenance medium to the cells that have grown into a monolayer, do not change the medium, and then place it in a 37°C, 5% CO2 incubator for culture.

[0131] The cells inoculated using these three different methods were frozen and thawed twice at -80°C, the supernatant was centrifuged to harvest the virus, and the titer was determined. This experiment was repeated three times, and the method with the highest titer was selected as the optimal inoculation method.

[0132] Results: CHN-YC and QR were inoculated on BHK-21 cells and Vero cells using three inoculation methods: synchronous inoculation, adsorption inoculation, and non-adsorption inoculation. The viruses were harvested three times. The results are shown in Tables 1 and 2. When CHN-YC was inoculated using adsorption inoculation, the virus titer was the highest, with an average titer of 10 7.2 TCID 50 / mL, so the best inoculation method for CHN-YC is adsorption inoculation. When QR is inoculated without adsorption, the virus titer is the highest, and the average titer can reach 10 6.0 TCID 50 / mL, so the best inoculation method for QR is non-adsorption inoculation.

[0133] Table 1 CHN-YC inoculation method screening

[0134]

[0135] Table 2 QR inoculation method screening

[0136]

[0137] Example 6 Selection of the best DTMUV virus species

[0138] 1. Duckling infection test

[0139] SPF duck embryos were purchased for incubation, and 102 SPF ducks were hatched. The ducks were raised to 5 days of age for the experiment, and the ducks were divided into an autopsy group and an observation group. The autopsy group was divided into 6 groups, with 8 ducks in each group, including the MC strain group, GA strain group, ZZ strain group, CHN-YC strain group, CHN-JL strain group and MOCK group. The observation group was also divided into 6 groups, with 9 ducks in each group, including the MC strain group, GA strain group, ZZ strain group, CHN-YC strain group, CHN-JL strain group and MOCK group. The MC strain group was injected with DTMUV MC strain virus through the leg muscle for 10 6 TCID 50 / 0.1mL, 1mL / feather; GA strain group injected DTMUVGA strain virus through leg muscle for 10 6 TCID 50 / 0.1mL, 1mL / feather; ZZ strain group injected DTMUV ZZ strain virus through leg muscle 10 6 TCID 50 / 0.1mL, 1mL / feather; CHN-YC strain group was injected with DTMUV CHN-YC strain virus through leg muscle for 10 6 TCID 50 / 0.1mL, 1mL / feather; CHN-JL strain group was injected with DTMUV CHN-JL strain virus through leg muscle for 10 6 TCID 50 The MOCK group received an intramuscular injection of cell culture medium (1 mL / bird) at 0.1 mL / day. The infection protocol is shown in Table 3.

[0140] Table 3DTMUV infection test plan

[0141]

[0142] The clinical manifestations and mental status of the 12 groups of ducklings were observed and recorded. Necropsy treatment of ducklings in the 12 groups: On days 3 and 7 post-infection, four ducklings from each group were randomly sacrificed. Necropsy changes were observed, and tissues and organs, including the heart, liver, spleen, brain, and kidney, were collected. Serum was collected, and images of the lesions were taken. Some organs were stored at -80°C for viral load analysis; others were immersed in 4% paraformaldehyde for HE staining and section preparation. Treatment of ducklings in the observation group: Daily weight was recorded for 1 to 14 days post-infection, and fecal swabs were collected and frozen at -80°C for testing of viral shedding patterns. All ducklings were sacrificed on day 14, and serum was collected for antibody testing.

[0143] 2. Treatment of virus infection test samples

[0144] The pathological tissues of animal experiments were collected. At 4°C, 3 to 5 volumes of DMEM medium and 100 μg / mL penicillin and 100 μg / mL streptomycin were added to the tissues. After being fully ground with a homogenizer, the tissue homogenate was placed in a -80°C refrigerator and frozen and thawed three times. Then, it was centrifuged at 12,000 rpm for 10 min at 4°C. The supernatant was collected and stored in a -80°C refrigerator for later use.

[0145] For fecal swabs, cover the cotton swab with PBS, vortex for 1 min to mix thoroughly, and then freeze-thaw three times at -80°C. Then, centrifuge at 12,000 rpm for 10 min at 4°C. Collect the supernatant and store in a -80°C refrigerator for later use.

[0146] 3. Viral Nucleic Acid Extraction and cDNA Synthesis

[0147] Viral DNA extraction Viral DNA was extracted using the TIANGEN viral DNA extraction kit. The method was referred to the instructions. The specific steps are as follows:

[0148] (1) The homogenate of the ground specimen was centrifuged at 12,000 rpm for 10 min at 4°C. The supernatant was discarded, and 200 μL of buffer GA was added. The suspension was vortexed until it was completely suspended.

[0149] (2) Add 20 μL of Proteinase K solution to the suspension, mix well, then add 200 μL of buffer GB, invert thoroughly to mix, and place at 70°C for 10 min. The solution should become clear. Centrifuge immediately and remove the water droplets on the inner wall of the tube cap.

[0150] (3) Add 200 μL of anhydrous ethanol to the tube and vortex thoroughly for 15 seconds. A flocculent precipitate may appear at this time, which should be centrifuged immediately. Add the solution and flocculent precipitate obtained in the previous step to an adsorption column CB3 and transfer them together to a collection tube. Centrifuge at 12,000 rpm for 30 seconds. Discard the waste liquid and return the adsorption column CB3 to the collection tube.

[0151] (4) Add 500 μL of buffer GD to the adsorption column CB3, centrifuge at 12,000 rpm for 30 seconds, discard the waste liquid, and return the adsorption column CB3 to the collection tube.

[0152] (5) Add 600 μL of rinse solution PW to the adsorption column CB3 and centrifuge at 12,000 rpm for 30 seconds. Discard the waste liquid and return the adsorption column CB3 to the collection tube. Repeat this operation, return the adsorption column CB3 to the collection tube, centrifuge at 12,000 rpm for 2 minutes, and discard the waste liquid.

[0153] (6) Place the adsorption column CB3 at room temperature for several minutes to completely dry the remaining rinse solution in the adsorption material. Transfer the adsorption column CB3 to a clean centrifuge tube and drop 50 μL of elution buffer TE onto the middle part of the adsorption membrane. Place the column at room temperature for 2-5 minutes. Centrifuge at 12,000 rpm for 2 minutes. Collect the solution into a centrifuge tube and store at -20°C for later use.

[0154] Viral RNA was extracted according to the Trizol instructions. The steps are as follows:

[0155] (1) Take 200 μL of the supernatant or serum of the diseased tissue and add it to a 1.5 mL enzyme-free centrifuge tube. Add 1 mL of Trizo to each tube to lyse the cells. Invert 10 to 20 times to mix well and let it stand at room temperature for 5 minutes.

[0156] (2) Add 200 μL of chloroform to the centrifuge tube, vortex for 15 seconds to mix thoroughly, place on ice for 10 minutes, allow the mixture to separate, and centrifuge at 4°C, 12,000 rpm for 5 minutes.

[0157] (3) Transfer 400 μL of supernatant to a new 1.5 mL enzyme-free centrifuge tube, add an equal volume of isopropanol, invert 10-20 times to mix, let stand at room temperature for 10 minutes, and centrifuge at 4°C, 12,000 rpm for 10 minutes to precipitate nucleic acids.

[0158] (4) Aspirate and discard the supernatant, add 1 mL of anhydrous ethanol for washing, centrifuge at 7500 rpm for 5 min at 4°C, discard the supernatant, centrifuge briefly and carefully discard the supernatant, and let the precipitate air dry at room temperature for 5-10 min.

[0159] (5) After the precipitate is completely dry, add an appropriate amount of DEPC water to each tube to dissolve it, and measure the A 260 and A 280 The RNA content and purity of the samples were determined and stored at -80℃ for future use.

[0160] Using the extracted RNA as a template, reverse transcription was performed to obtain cDNA. The reverse transcription system was as follows:

[0161]

[0162] Heat at 65°C for 5 min, quickly cool on ice, and let stand on ice for 2 min.

[0163]

[0164] Mix gently by pipetting, incubate at 42°C for 2 min, and then place on ice.

[0165]

[0166] Use a pipette to gently blow and mix, 25℃5min, 50℃45min, 85℃2min, 4℃∞,

[0167] The synthesized cDNA was stored at -20°C for future use.

[0168] 4. Detection of viral load in animal test samples

[0169] (1) Extract total RNA from various tissues, organs, and serum according to the method in 3.2.9.2, and determine the concentration and purity of RNA. Reverse transcribe RNA into cDNA according to the instructions of the Novozyme reverse transcription kit. The specific reaction is as follows:

[0170]

[0171] Mix gently by pipetting and incubate at 42°C for 2 min.

[0172]

[0173] Mix gently by pipetting, incubate at 50°C for 15 min, then at 85°C for 5 sec. After the reaction is complete, store at 20°C.

[0174] (2) The reversed cDNA samples were amplified using AceQ qPCR SYBRGreen Master Mix, a premixed reagent containing ROX from Norvegian, in an ABI ViiA 7 fluorescence quantitative instrument. The fluorescence quantitative primers for DTMUV were designed based on the DTMUV E protein gene published in GenBank, and the fluorescence quantitative primers for NDRV were designed based on the NDRVσC protein gene published in GenBank. The primer base sequences are shown in Table 4.

[0175] Table 4 qPCR primer sequences

[0176]

[0177] The primers were synthesized by Wuhan Qingke Biotechnology Co., Ltd. and dissolved in DEPC water according to the instructions, configured to the storage concentration, and stored at -20°C for future use.

[0178] Take the plasmid standard, dilute it 10 times, and use it as the qPCR template for RT-qPCR. 1 ~10 10 The positive plasmid copies / μL was plotted logarithmically to form a standard curve and the corresponding regression equation was calculated. The reaction system and reaction procedure are as follows:

[0179]

[0180]

[0181] Fluorescence quantitative PCR reaction system (20 μL) was used. The reaction conditions were: 95°C for 5 minutes of pre-denaturation, followed by 40 cycles of 95°C for 10 seconds and 60°C for 30 seconds. Fluorescence signal changes throughout the reaction were detected using an ABIPrism 7500 real-time fluorescence quantitative PCR instrument, and the resulting data were calculated and analyzed.

[0182] 5. Preparation of HE Sections of Animal Experimental Samples

[0183] (1) Dehydration. After 24 hours, remove the immersed tissue from the 10% formalin solution, rinse twice with clean deionized water, and then place it in 70% ethanol to complete the dehydration process.

[0184] (2) Transparency. Immerse the tissue in a mixture of anhydrous ethanol and xylene (1:1) for 1.5 hours, then immerse it in xylene until the tissue becomes transparent.

[0185] (3) Wax immersion and embedding. Take out the transparent tissue and place it in melted paraffin wax for immersion. Then place the wax-immersed tissue in melted solid paraffin wax and wait for it to solidify into a wax block containing the tissue. This is embedding.

[0186] (4) Slice and bake. After trimming the wax block slightly, place it on a microtome and cut it into several 4-6 μm wax strips. After flattening it on a warm water surface, place it on a glass slide and bake it in a 60°C incubator for 30 minutes.

[0187] (5) Staining. The dried sections were dewaxed with xylene, debenzened step by step with different concentrations of alcohol, rehydrated with distilled water, and then stained with HE. The stained sections were dehydrated with alcohol gradients and made transparent with xylene.

[0188] (6) Covering. Add a drop of neutral gum to the center of the slice, slowly lower the cover slip, and then observe the prepared slice under a normal microscope.

[0189] 6. Antibody detection in animal serum

[0190] The blocking ELISA method is used to detect antibodies in the serum. Anti-DTMUV serum inhibition rate > 14.659% can be determined as positive, and anti-NDRV serum inhibition rate > 16.9% can be determined as positive. The operation steps are as follows:

[0191] (1) Coating: DTMUV EⅢ protein and NDRVσC protein diluted to 0.25 μg / mL were added to a 96-well ELISA plate at 100 μL / well and coated overnight at 4°C.

[0192] (2) Washing: Add 200 μL / well of prepared PBS-T and wash three times, with an interval of about 5 minutes between each wash.

[0193] (3) Blocking: Add 200 μL of 1% BSA as blocking solution to each well, block in a 37°C constant temperature incubator for 1 hour, and then wash three times with PBS-T.

[0194] (4) Primary antibody incubation: The serum to be tested was diluted with PBS-T at a ratio of 1:20, and 100 μL / well was added to the ELISA plate. Positive and negative serum controls and four blank control wells with only the diluent were set up. After incubation at 37°C in a constant temperature incubator for 1 h, the plates were washed three times with PBS-T.

[0195] (5) Monoclonal antibody incubation: Dilute the purified monoclonal antibody ascites to 0.5 μg / mL and add it to the ELISA plate at 100 μL / well. Add the monoclonal antibody to 2 of the 4 blank wells, and only add the diluent to the other 2 wells. Incubate in a 37°C constant temperature incubator for 1 hour, and then wash 3 times with PBS-T.

[0196] (6) Secondary antibody incubation: Horseradish peroxidase (HRP)-labeled goat anti-mouse IgG was diluted 1:7500 and added to the ELISA plate at 100 μL / well. The plate was incubated in a 37°C constant temperature incubator for 1 h and then washed three times with PBS-T.

[0197] (7) Color development: Add TMB color development solution to the ELISA plate at 50 μL / well and incubate in the dark at 37°C for 5 min.

[0198] (8) Termination: Add 2M H2SO4 to the ELISA plate at 50 μL / well to terminate the reaction.

[0199] (9) Reading: After stopping the reaction, measure the OD in a microplate reader. 450nm The value of .

[0200] (10) Calculation results: According to the formula, inhibition rate (IP) = (negative OD 450nm Value - Sample OD 450nm value) / (negative OD 450nm value)×100%.

[0201] 7. Results:

[0202] (1) Clinical symptoms, weight gain and survival rate of ducks in each group after virus infection

[0203] After inoculating ducklings with different strains of DTMUV, the ducklings were observed for 14 days. Symptoms began to appear in each group 3 days after infection. The sick ducks had a poor appetite, were depressed, and excreted green loose feces. Among them, the symptoms of the CHN-YC strain group were the most obvious. The ducklings showed symptoms of sitting or lying down, tilted head and neck, closed eyes, twitching legs and feet, and unstable standing and walking. Some ducklings showed neurological symptoms and opisthotonos. The specific symptoms are shown in Figure 7 .

[0204] The weight changes of ducks in different groups are as follows Figure 8 -A shows that the weight gain of ducklings in the MOCK group was normal, while the weight gain of each infected group slowed down significantly. The weight gain of the GA and ZZ strain groups was relatively slow from 1 to 7 days after infection, but after 7 days, the ducklings had survived and the weight gain rate was similar to that of the MOCK group. The weight gain rate of the MC and CHN-JL strain groups was slower than that of the GA and ZZ strain groups. The weight of the MC strain group had almost no weight gain 6 days after infection, and then the weight began to increase slowly. The weight gain of the CHN-YC strain group was the slowest, and there was no significant increase in weight until the 8th day after infection. The weight only began to increase slowly after 8 days, and the growth rate was only half of that of the ducklings in the MOCK group. Survival rate is shown in Figure 1-A. Figure 8 As shown in Figure 3-B, only the CHN-YC and MC strain groups experienced duckling mortality. The CHN-YC strain-inoculated ducklings experienced two deaths on day 4, two on day 5, and one on day 6, resulting in a total mortality rate of 55%, the highest among all infected groups. In contrast, the MC strain-inoculated ducklings experienced one death on day 6 and one on day 7, for a total mortality rate of 22%. No ducklings in the other infected groups or the MOCK group died within 14 days of infection.

[0205] (2) Detoxification of ducks in each group after infection

[0206] The viral load in the fecal swabs of infected ducks in each group was detected using RT-qPCR. Figure 9 The virus could be detected in fecal swabs of each group on the first day after infection. Among the five groups, the viral load of fecal swabs in the CHN-YC strain group was the highest. The two peaks of virus excretion in the CHN-YC strain group, the MC strain group, the GA strain group, and the ZZ strain group were on the 5th and 11th days after infection, while the two peaks of virus excretion in the CHN-JL strain group were on the 6th and 11th days after infection. Subsequently, the amount of virus excretion in each group gradually decreased, and on the 14th day, the ducks in each group could still be detected to be excreting the virus.

[0207] (3) Necropsy lesions and histopathological changes in ducks of each group after infection

[0208] After infection, the ducklings in the autopsy group were dissected, and it was found that the organs of each infection group had different degrees of lesions. Figure 10 The shape, size and luster of the organs of ducks in the MOCK group were normal. Figure 10As shown in -A, the spleens of the CHN-YC and MC strain groups had white necrotic foci and were swollen, resembling a "mottled spleen." The CHN-YC strain group had the most obvious spleen enlargement. The spleens of the CHN-JL strain ducklings were mainly swollen due to hemorrhage, and the spleen color became darker. The spleens of the ZZ and GA strain ducklings were slightly enlarged, with hemorrhagic and necrotic spots. Figure 10 As shown in Figure 3-B, the brain lesions in the CHN-YC strain group were the most severe, with swelling and dendritic hemorrhages in the cerebral blood vessels. The dendritic hemorrhages in the cerebral blood vessels in the MC strain group were also more serious. The brains of the other infection groups also had swelling and hemorrhages, but the lesions were milder. Figure 10 As shown in Figure 3-C, the heart of the CHN-YC strain group had a large hemorrhage focus and was swollen. The heart of the CHN-JL strain group was swollen and soft. The heart of the ZZ strain group was slightly swollen but no hemorrhage focus was found. One hemorrhage spot was found in the heart of the GA strain group. The heart of the MC strain group was slightly swollen and had a hemorrhage spot. Figure 10 -D, the livers of the CHN-YC strain group had bleeding foci, brittle texture, blunt edges, necrotic swelling, while the livers of the CHN-JL strain group and the MC strain group were more obviously swollen, and the livers of the CHN-JL strain group turned into khaki. The main changes in the livers of the GA strain group and the ZZ strain group were severe bleeding and slight swelling.

[0209] On the 7th day after infection, the spleen, liver and brain of the ducklings in the autopsy group were collected and pathological sections were made. The pathological histological changes of the organs in each group were observed. Figure 11 From 11-A, it can be seen that the spleen of the infected group showed congestion and lymphocyte necrosis, and the spleen of the CHN-YC strain group also showed macrophage proliferation and necrosis; Figure 11 -B liver pathological sections showed focal necrosis and lymphocytic infiltration in the livers of the CHN-YC and MC strain groups, hepatic hemorrhage and focal necrosis in the ZZ strain group, and vacuolar degeneration in the livers of the GA and CHN-JL strain groups; Figure 11 Brain pathological sections of the CHN-YC strain showed varying degrees of vascular cuffing and satellite formation, as well as focal necrosis, in all groups. Observation of multiple sections revealed the most severe histopathological changes in organs of the CHN-YC strain group.

[0210] (4) Detection of viral load in organs of ducks in each group after infection

[0211] The viral load of the organs of ducks in each group was detected by RT-qPCR on the 3rd and 7th day after infection. Figure 12. On the 3rd and 7th days after infection, the virus could be detected in the organs and serum of ducks in all infection groups, with the highest viral load in the spleen, indicating that the spleen is the main target organ of DTMUV after infection. Except for the lower viral load in the spleen on the 3rd day of infection and the viral load in the liver on the 7th day of infection in the CHN-YC strain group than in the MC strain group, the viral load in each organ was the highest at other times, and the viral load in the brain of the CHN-YC strain group was much higher than that of other groups. The viral load in the heart, serum, kidney and liver of the CHN-YC strain ducklings was the highest on the 3rd day of infection. There was no significant change in the viral load in the spleen on the 3rd and 7th days of infection, and the viral load in the brain was the highest on the 7th day of infection. In the other infection groups, the viral load in each organ and serum of the ducklings was the highest on the 3rd day, and then decreased on the 7th day.

[0212] (5) Antibody detection in duck serum of each group after infection

[0213] Blood was collected from each group of ducklings 14 days after infection to prepare serum, and the antibody level was detected using the blocking ELISA method. Figure 13 Fourteen days after infection, antibodies were detected in the sera of ducks from all infected groups, except the ZZ and CHN-JL strain groups. The highest antibody level in the CHN-YC strain group reached 45.7%, and all ducklings in this group tested positive. The serum test positivity rate in the MC strain group was 75%, with a maximum antibody level of 24.1%. The serum test positivity rate in the GA strain group was 50%, with a maximum antibody level of 20.5%.

[0214] Example 6 Optimization of Preparation Conditions for a Combined Inactivated Vaccine of Duck Tembusu Virus and New Duck Reovirus

[0215] 1. Large-scale preparation of virus seed and TCID 50 Determination of

[0216] (1) The CHN-YC strain of duck Tembusu virus was inoculated on BHK-21 cells at a dose of 0.01 to 0.1 MOI by adsorption inoculation. The virus was collected 48 to 60 hours after inoculation. The virus titer (TCID 50 ),

[0217] (2) The novel duck reovirus QR-China / 2020 strain was inoculated on Vero cells at a dose of 0.1-0.2 MOI using a non-adsorption inoculation method, and the virus was collected 72-96 hours after inoculation. After the virus was collected, the virus titer (TCID 50 ) determination.

[0218] Results: CHN-YC and QR viruses were inoculated into cells and the virus liquid was harvested and purified by virus plaque assay. 100 mL of virus liquid was prepared for each virus, and the TCID of CHN-YC was measured. 50 is 10 -6.7 / 0.1mL, QR TCID 50 is 10 -6.1 / 0.1mL.

[0219] 2. Sterility testing of virus strains

[0220] Take 200 μL of CHN-YC and QR virus liquid and inoculate them into LB medium, FTM medium and TSB medium respectively. Inoculate Streptococcus suis into these three culture media as a control. Culture them at 37°C for 7 days and observe the changes in the culture medium every day.

[0221] Results: The virus was inoculated into different bacterial culture media for sterility testing. The results are shown in Table 5. After the two virus liquids were inoculated into the bacterial culture media, the culture media were clear and no turbidity appeared. However, after the positive control was inoculated with Streptococcus suis, the bacterial culture media became turbid. Therefore, it can be determined that there was no bacterial and fungal contamination in the two virus liquids.

[0222] Table 5 Sterility test results of various viruses

[0223]

[0224] Note: + represents turbidity of the culture medium, - represents clear culture medium

[0225] 3. Detection of exogenous viruses in virus strains

[0226] The virus liquid of CHN-YC and QR was taken, and RNA was extracted and reverse transcribed into cDNA and DNA was extracted according to the above method. The virus liquid was subjected to PCR detection for Marek's disease virus (MDV), duck paramyxovirus (DPMV), Muscovy duck reovirus (MDRV), duck hepatitis A virus (DHAV), egg drop syndrome virus (EDSV), duck adenovirus type 3 (DAdV-3) and Newcastle disease virus (NDV) according to the PCR primer sequences in Table 6.

[0227] Table 6 PCR primer sequences

[0228]

[0229] The PCR reaction system and reaction procedure are as follows:

[0230]

[0231] The total PCR reaction volume was 50 μL. The amplification conditions were: 95°C for 3 min of initial denaturation, followed by 35 cycles of 95°C for 15 sec, 60°C for 15 sec, and 72°C for 60 sec / kb, followed by an extension at 72°C for 10 min.

[0232] Results: After reverse transcription or DNA extraction from the virus solution, PCR was performed to detect exogenous viruses. The results were as follows: Figure 14 PCR detection of MDV, DPMV, MDRV, DHAV, EDSV, DAdV-3, and NDV in CHN-YC and QR was negative, indicating that there was no exogenous virus contamination in these two virus solutions.

[0233] 4. Optimization of the best inactivation conditions for virus solution

[0234] Formaldehyde was added to the CHN-YC and QR virus solutions at final concentrations of 0.5‰, 1‰, 1.5‰, and 2‰, respectively, and stirred thoroughly. The mixture was then transferred to a fresh container and divided into three groups for each concentration. The mixture was shaken at 110 rpm at 37°C. The inactivation time for the three groups was set to 12 hours, 24 hours, and 36 hours, respectively. The inactivated CHN-YC and QR virus solutions were inoculated into BHK-21 and Vero cells that had grown to monolayers. Cell growth was observed daily to observe whether the cells developed lesions. The virus solution was blindly passaged for three generations, and the cells were observed under a microscope at each generation for the presence of lesions. If no lesions were observed for three generations, the virus was considered completely inactivated.

[0235] Results: Different inactivation agent concentrations and inactivation times were used to inactivate the virus, and inactivation tests were performed. The results are shown in Tables 7 and 8. To minimize vaccine side effects, the optimal inactivation conditions should be those that achieve complete viral inactivation with the lowest inactivation agent concentration and shortest inactivation time. When inactivating CHN-YC, the lowest formaldehyde concentration was 1‰. After inactivation at 37°C for 24 hours, cells were inoculated onto cells and blindly passaged for three passages without exhibiting pathological changes. Therefore, the optimal inactivation conditions for CHN-YC were a formaldehyde concentration of 1‰ and an inactivation at 37°C for 24 hours. When inactivating QR, the lowest formaldehyde concentration was 1.5‰. After inactivation at 37°C for 24 hours, cells were inoculated onto cells and blindly passaged for three passages without exhibiting pathological changes. Therefore, the optimal inactivation conditions for NDRV were a formaldehyde concentration of 1.5‰ and an inactivation at 37°C for 24 hours.

[0236] Table 7 CHN-YC inactivation test results

[0237]

[0238] Note: + represents cells with CPE, - represents cells without CPE

[0239] Table 8 QR inactivation test results

[0240]

[0241] Note: "+" represents cells with CPE, "-" represents cells without CPE

[0242] Example 7

[0243] Duck Tembusu virus and new duck reovirus dual inactivated vaccine, which includes inactivated duck Tembusu virus and new duck reovirus, among which the content of duck Tembusu virus CHN-YC is 10 7.7 TCID 50 / mL, the content of novel duck reovirus QR was 10 7.1 TCID 50 / mL, and the volume ratio of duck Tembusu virus CHN-YC to novel duck reovirus QR was 1:1.

[0244] The preparation method of the above-mentioned duck Tembusu virus and novel duck reovirus combined inactivated vaccine comprises the following steps:

[0245] Prepare the oil phase at a ratio of 94 parts medical white oil to 6 parts Tween-80, then add 2 parts aluminum stearate, mix thoroughly, and sterilize by autoclave at 121°C for 15 minutes. Cool to room temperature and set aside. Separately, take 96 parts of a 1:1 mixed antigen solution that has been inactivated and passed the inspection, add 4 parts of Tween-80 that has been autoclaved at 121°C for 15 minutes, and stir evenly to prepare the aqueous phase. Mix the oil phase and aqueous phase at a ratio of 2:1 (V / V). Add the oil phase in a high-speed emulsifier and stir slowly while slowly adding the aqueous phase. After addition, stir at 10,000 rpm for 5-15 minutes. After aliquoting, store at 4°C.

[0246] Example 8: Testing of the properties of the combined inactivated vaccine of duck Tembusu virus and new duck reovirus

[0247] 1. Methods

[0248] (1) Appearance observation and dosage form test: Observe the appearance of the inactivated vaccine with the naked eye to check for the presence of impurities such as precipitation. Add the prepared vaccine drop by drop onto the surface of cold water and observe the diffusion of the remaining drops except the first drop. If the remaining drops do not diffuse, it indicates that the vaccine dosage form is water-in-oil.

[0249] (2) Stability test: Take 10 mL of vaccine sample and centrifuge at 3000 r / min for 15 min. Observe whether the vaccine has stratification. There should be no demulsification, stratification or precipitation. The aqueous phase precipitated at the bottom of the tube should be ≤0.2 mL.

[0250] (3) Viscosity measurement: At 20-25°C, take 1 mL of vaccine and allow 0.4 mL of vaccine to flow out naturally vertically from an outlet with an inner diameter of 1.2 mm. Repeat this three times. The time consumed in this process should be within 2-8 seconds.

[0251] (4) Shelf life test: Take 20 mL of the developed oil-emulsion inactivated vaccine, divide it into two equal parts, and put them into two 10 mL centrifuge tubes respectively. Place the two tubes at 4°C and 37°C respectively, and observe continuously. Check monthly whether the vaccine shows any changes such as stratification and demulsification.

[0252] (5) Sterility test: The developed oil-emulsion inactivated vaccine was inoculated into LB medium, FTM medium and TSB medium, and Streptococcus suis was inoculated into these three culture media as a control. The culture media were placed in a 37°C environment for 7 days, and the changes in the culture medium were observed every day.

[0253] 2. Results:

[0254] The experiment prepared a combined inactivated vaccine of DTMUV and NDRV and tested the properties of the vaccine.

[0255] Properties: Milky white uniform emulsion;

[0256] Dosage form: Oil-in-water type. Take a clean pipette and draw a small amount of vaccine onto the surface of cold water. Except for the first drop, it will not spread.

[0257] Stability: Pipette 10 mL of vaccine into a centrifuge tube and centrifuge at 3000 rpm / min for 15 min. No demulsification, stratification or precipitation occurs, and the aqueous phase precipitated at the bottom of the tube is ≤0.2 mL.

[0258] Viscosity: At room temperature, the time taken for a 1 mL pipette to vertically dispense 0.4 mL of vaccine three times was 5.4 s, 4.8 s, and 4.6 s, respectively, which meets the viscosity requirements of oil-emulsion inactivated vaccines (2-8 s).

[0259] Sterility test of vaccine: The vaccine was inoculated into different bacterial culture media for sterility test. The results are shown in Table 9. Seven days after vaccination, the bacterial culture medium was still not turbid, while the positive control group showed obvious turbidity on the third day after vaccination, indicating that the vaccine had no bacterial and mold contamination and passed the sterility test.

[0260] Table 9 Vaccine sterility test results

[0261]

[0262] Note: + represents turbidity of the culture medium, - represents clear culture medium

[0263] The prepared vaccine was placed in 37°C and 4°C environments, respectively, and observed monthly for demulsification, denaturation, and precipitation. The results are shown in Table 10. When the vaccine was stored at 37°C, demulsification and stratification occurred in the second month, while when the vaccine was stored at 4°C, no deterioration was observed in the seventh month. Therefore, the vaccine can be stored for one month at 37°C and for at least seven months at 4°C.

[0264] Table 10 Vaccine shelf life test

[0265]

[0266] Note: “-” indicates that the vaccine is stable and not stratified, and “+” indicates that the vaccine is stratified or demulsified.

[0267] Example 9 Safety Test of the Combined Inactivated Vaccine of Duck Tembusu Virus and New Duck Reovirus

[0268] 1. Methods

[0269] According to the selected viruses and inactivation conditions, inactivated vaccines were prepared and safety tests were carried out. The vaccines were used to inoculate SPF ducks with a single dose, repeated single doses, double doses, and repeated double doses according to the following steps, wherein:

[0270] (1) Single-dose single vaccination group: Ten 7-day-old SPF ducks were injected intramuscularly with the vaccine at 0.5 mL / bird. Another 10 7-day-old SPF ducks were used as the non-immunized control group. The ducks were observed for 14 consecutive days after injection. The mental state, water intake, and food intake of each group were observed daily. The weights of the ducks in the vaccinated and control groups were weighed daily and recorded. After immunization, the injection site of each immunized duck was palpated to check for local injection reactions such as redness and swelling. All ducks were sacrificed on day 14 to inspect the vaccine absorption at the injection site and observe for organ lesions.

[0271] (2) Double-dose single vaccination group: Ten 7-day-old SPF ducks were injected intramuscularly with the vaccine (1 mL / bird). Another 10 7-day-old SPF ducks were used as the non-immunized control group. The ducks were observed for 14 consecutive days after injection. The mental state, water intake, and food intake of each group were observed daily. The weights of the ducks in the vaccination and control groups were weighed daily and recorded. After immunization, the injection site of each immunized duck was palpated to check for local injection reactions such as redness and swelling. All ducks were sacrificed on day 14 to inspect the vaccine absorption at the injection site and observe for organ lesions.

[0272] (3) Single-dose repeated vaccination group: Ten 7-day-old SPF ducks were injected intramuscularly with the vaccine at 0.5 mL / bird. The same dose was repeated 14 days later. Another 10 7-day-old SPF ducks were used as the non-immunized control group. After the two injections, the ducks were observed for 14 consecutive days. The mental state, water intake, and food intake of each group were observed daily. The weights of the ducks in the vaccinated and control groups were weighed daily and recorded. After immunization, the injection site of each immunized duck was palpated to check for local injection reactions such as redness and swelling. All ducks were sacrificed after 14 days of observation to check for vaccine absorption at the injection site and to observe for organ lesions.

[0273] (4) Double-dose repeated vaccination group: Ten 7-day-old SPF ducks were injected intramuscularly with the vaccine (1 mL / bird) and the same dose was repeated 14 days later. Another 10 7-day-old SPF ducks were used as the non-immunized control group. After the two injections, the ducks were observed for 14 consecutive days. The mental state, water intake, and food intake of each group were observed daily. The weights of the ducks in the vaccinated and control groups were weighed daily and recorded. After immunization, the injection site of each immunized duck was palpated to check for local injection reactions such as redness and swelling. All ducks were sacrificed after 14 days of observation to check for vaccine absorption at the injection site and to observe for organ lesions.

[0274] 2. Conclusion

[0275] When conducting a single immunization safety test, the ducklings in the single-dose group were injected with 0.5 mL of vaccine into the leg muscle, and the ducklings in the double-dose group were injected with 1 mL of vaccine into the leg muscle. The ducklings were observed for 14 days after immunization. The mental state and weight data of the ducks in each group can be seen in Table 11. The autopsy results can be seen in Table 11. Figure 15 After vaccination, the immunized ducklings showed a slight lethargy on the first day, which returned to normal on the second day. SPSS software compared the daily weight gain between the immunized and control groups and revealed no significant difference. After vaccination, the ducklings were touched at the inoculation site and no swelling was observed. Autopsies revealed normal organ morphology and color in the immunized ducklings, with no vaccine fluid remaining at the inoculation site.

[0276] Table 11 Vaccine single immunization safety test

[0277]

[0278]

[0279] When repeat immunization safety test was conducted, ducklings in the single-dose group were injected with 0.5 mL of vaccine into the leg muscle, and then immunized again with the same dose 14 days later. Ducklings in the double-dose group were injected with 1 mL of vaccine into the leg muscle, and then immunized again with the same dose 14 days later. The ducklings were observed for 14 consecutive days after immunization. The mental state and weight data of the ducks in each group can be seen in Table 12, and the autopsy results can be seen in Table 12. Figure 16SPSS software was used to compare the daily weight gain of the immunized and control groups, revealing no significant difference. After vaccination, the inoculation site of the ducklings was touched and no swelling was observed. Autopsies revealed that the organs of the immunized ducklings were normal in morphology and color, with no vaccine fluid remaining at the inoculation site.

[0280] Table 12 Vaccine Repeated Immunization Safety Test

[0281]

[0282] Example 10 Safety and effectiveness testing of a combined inactivated vaccine of duck Tembusu virus and new duck reovirus

[0283] 1. Determination of serum neutralizing antibody levels

[0284] (1) Cell preparation: Take well-grown BHK-21 cells and Vero cells, discard the culture medium, wash twice with PBS, add trypsin for digestion, count the cells, dilute them to the required concentration with cell maintenance medium, and place them in an incubator for later use.

[0285] (2) Serum preparation: All serum samples need to be inactivated in a 56°C water bath for 30 min before use.

[0286] (3) Add 50 μL of inactivated serum to the first row of an empty 96-well plate, then dilute the serum with the virus inoculum in a 2-fold dilution ratio to the last row, and discard 50 μL of liquid from the last row.

[0287] (4) Dilute the virus to 200 TCID 50 , add 50 μL to each well of the above 96-well plate. At the same time, set up the serum toxicity control to be tested, negative and positive serum controls, virus control and normal cell control, of which 200 TCID 50 , 20 TCID 50 , 2 TCID 50 , 0.2 TCID 50 4 different concentrations.

[0288] (5) Place the 96-well cell culture plate in a 37°C, 5% CO2 incubator for 45 to 60 minutes.

[0289] (6) Add 100 μL / well of cell suspension to a 96-well plate, culture in a 37°C, 5% CO2 incubator, and observe the cell status every day.

[0290] (7) Result determination: Observe whether the cells have pathological changes and calculate the neutralizing antibody titer in the serum according to the Reed-Muench method. When the neutralization titer in the serum is ≥3log2, it is determined to be positive; when the neutralization titer in the serum is <3log2, it is determined to be positive.

[0291] (8) Validity of results: If the cells in the normal cell control wells fail to form a monolayer, die, or are contaminated with bacteria or fungi, or if CPE occurs in the uninoculated control cells, the results will be considered invalid.

[0292] 2. Determination of the minimum immunizing dose of the vaccine

[0293] Forty-eight healthy 7-day-old SPF ducklings were randomly divided into 4 groups, with 12 ducks in each group. Groups 1 to 3 received intramuscular injections of the prepared DTMUV and NDRV dual inactivated vaccine at three immunization doses of 0.2 mL, 0.5 mL, and 1 mL, respectively, and a booster immunization was performed 2 weeks later. The 12 ducks in the control group received intramuscular injections of sterile PBS in the leg according to the same immunization procedure.

[0294] One week after the first vaccination, blood was collected from 10 ducks in each group every week and serum was separated. Antibody levels in the ducks were tested by blocking ELISA and virus neutralization test until two weeks after the second vaccination. The antibody levels of each group were compared. Two weeks after the second vaccination, each group was challenged with the virus. Twelve ducks were taken from each of the experimental and control groups, and 6 ducks from each group were injected with 10 7 TCID 50 The DTMUV virus liquid and NDRV virus liquid were used to test the ducks. The weight of each group of ducks was weighed every day and the data was recorded for comparison. Blood samples were collected on the first and second days after the challenge, and the serum was separated and the virus content in the serum was detected. The challenge protection rate of each group of experimental ducks was calculated.

[0295] The minimum immune dose of the vaccine was determined by comparing the antibody levels and virus protection rates of each group.

[0296] 3. Determination of the duration of vaccine immunity

[0297] Thirty 7-day-old SPF ducks were randomly divided into three groups, with 10 ducklings in each group. Group 1 was a single immunization group, and each duck was injected with 0.5 mL of vaccine into the leg muscle. Group 2 was a repeated immunization group, and the vaccine was injected in the same way, 0.5 mL / bird, and a booster immunization was performed with the same dose 2 weeks later. Group 3 was a control group, and sterile PBS was injected into the leg muscle, 0.5 mL / bird.

[0298] Blood was collected before and after immunization every week. The collected whole blood was placed in a 37°C incubator for 2 hours, centrifuged at 4000 rpm / min for 5 minutes, and the upper serum was drawn into a new EP tube, marked, and placed in a -20°C refrigerator for later use.

[0299] Serum neutralization test and blocking ELISA were used to detect anti-DTMUV and anti-NDRV antibodies in the serum, and the antibody titer change curve in the serum after immunization was drawn based on the antibody titer obtained from the serum test.

[0300] 4. Results:

[0301] Seven-day-old ducklings were immunized with different doses of the vaccine and the antibody levels in the duck serum were measured 4 weeks after immunization using blocking ELISA and serum neutralization test. Figure 17 As shown in the figure: By detecting anti-DTMUV antibodies in the serum of each vaccine group through blocking ELISA, it was found that the serum antibody positivity rate of the 0.2mL vaccine immunization group did not reach 100% at the fourth week after immunization, but was only 60%, while the antibody positivity rate of the 0.5mL vaccine immunization group and the 1mL vaccine immunization group in the fourth week after immunization reached 100%, among which the serum inhibition rate of the 0.5mL vaccine immunization group was the highest, reaching 47.1%. Figure 17 -B. The anti-DTMUV neutralizing antibodies in the serum of the vaccine group were determined through serum neutralization test. It was found that the neutralizing antibody titer of the 0.2mL vaccine immunization group was still low at the fourth week after immunization, while the serum neutralizing antibody titer of the 0.5mL vaccine immunization group and the 1mL vaccine immunization group could reach 3log2 or above at the fourth week after immunization, and the neutralization antibody positivity rate reached 100%.

[0302] like Figure 17 -C, by blocking ELISA detection of anti-NDRV antibodies in the serum of each vaccine group, it was found that the antibody positivity rate of the 0.2mL vaccine immunization group had not reached 100% at the 4th week after immunization, and the antibody positivity rate and antibody titer were both low, while the antibody positivity rate in the serum of the 0.5mL vaccine immunization group and the 1mL vaccine immunization group both reached 100%, and the antibody level in the serum of the 1mL vaccine immunization group was relatively higher. Figure 17 -D, and then the neutralizing antibody level was tested through serum neutralization test, and it was found that the neutralizing antibody level continued to rise with the extension of time and the increase of dosage. The neutralizing antibody positivity rate in the serum of the 0.5mL vaccine immunization group and the 1mL vaccine immunization group could reach 100% in the 4th week, and the neutralizing antibody level in the ducks could reach as high as 5log2.

[0303] Then, each vaccine group and the control group were challenged with DTMUV and NDRV, and the body weight was measured every day and the serum was collected for viral load detection. Figure 18 and Figure 19 As shown. After the challenge, viruses were detected in the serum of the ducks in the challenge group that were not vaccinated. The detection rate of DTMUV in the serum of the 0.2mL vaccine immunization group was 33.3%, and the detection rate of NDRV was 50.0%. However, DTMUV and NDRV were not detected in the serum of the ducklings in the 0.5mL and 1mL vaccine immunization groups. Figure 19It can be found that the weight gain rate of the unvaccinated challenged group began to slow down on the second day after the challenge, and the daily weight gain continued to decline. On the fifth day, the weight began to gradually recover. The weight change in the 0.2mL vaccine immunization group was better than that of the unvaccinated challenged group, but it was still lower than the net daily weight gain of the MOCK group. The weight gain of ducks in the 0.5mL and 1mL vaccine immunization groups had no significant difference from that of the MOCK group, indicating that when the vaccine immunization dose is 0.5mL / feather and 1mL / feather, it has a good immune protection effect on ducks and can provide 100% protection.

[0304] Example 11 Determination of the duration of vaccine immunity for safety testing of a dual inactivated duck Tembusu virus and novel duck reovirus vaccine

[0305] The serum antibody levels of the single vaccine immunization group and the repeated vaccine immunization group were continuously tested. The anti-DTMUV antibody levels in the serum of the single immunization 0.5 mL vaccine group were Figure 20 It was found that when only a single immunization was performed, the antibody level in the serum reached a high level in the third week, at which time the antibody positivity rate was 90%. Subsequently, the antibody level slowly decreased over time until it dropped to a lower level in the eighth week. Through serum neutralization test, it was found that the neutralizing antibody level in the serum also changed similarly to the antibody level. The neutralizing antibody level was the highest in the third week, reaching 5log2.

[0306] The anti-DTMUV antibody levels in the serum of the repeated immunization 0.5 mL vaccine group were as follows: Figure 21 7-A shows that after repeated immunization, serum antibody levels reached their highest level at week 7, with an antibody positivity rate of 100% and a serum suppression rate of up to 72.1%. Antibodies were still detectable in the serum of most ducks until week 12. 1-7-B also shows that serum neutralizing antibody levels peaked at week 7, reaching 9 log2, demonstrating excellent protection against the virus.

[0307] The anti-NDRV antibody levels in the serum of the single immunization 0.5 mL vaccine group were as follows: Figure 22 The antibody level in ducks was the highest in the third week after the first vaccination, with an antibody positivity rate of 70%, and then gradually decreased. The neutralizing antibody level in the serum could reach a maximum of 4log2 at the third week, and then decreased. By the seventh week, no antibodies could be detected in the serum.

[0308] The anti-NDRV antibody levels in the serum of the repeated immunization 0.5 mL vaccine group were as follows: Figure 23 The antibody positivity rate reached 100% at the 4th week, and the antibody and neutralizing antibody levels in the serum rose to the highest point at the 6th week. The serum suppression rate could reach more than 60% at the 6th week, and the neutralizing antibody level could reach 6log2, which was higher than the antibody level of the single immunization group and lasted longer.

[0309] In summary, it can be found that repeated immunization has higher antibody levels and longer duration of immunity than single immunization. When repeated immunization is carried out, antibodies can still be detected in the serum at week 12. A second immunization two weeks after the first immunization is the most suitable immunization method for ducks with this vaccine.

[0310] Although the above embodiments have been described in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments based on this embodiment without inventiveness, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A Duck Tembusu virus CHN-YC, deposited with CCTCC NO: V202371.

2. Use of the duck Tembusu virus CHN-YC according to claim 1 in preparing a vaccine for preventing and treating duck Tembusu virus.

3. A combined inactivated vaccine of duck Tembusu virus and novel duck reovirus, characterized in that: The bivalent inactivated vaccine includes inactivated duck Tembusu virus CHN-YC and inactivated novel duck reovirus QR-China / 2020; wherein, the preservation number of the duck Tembusu virus CHN-YC is: CCTCC NO: V202371; the preservation number of the novel duck reovirus (NovelDuckReovirus) QR-China / 2020 is: CCTCC NO: V202372.

4. The duck Tembusu virus and novel duck reovirus combined inactivated vaccine according to claim 3, characterized in that: In the bivalent inactivated vaccine, The content of duck Tembusu virus CHN-YC is 10 7 ~10 8 TCID 50 / mL, The content of the new duck reovirus QR-China / 2020 is 10 7 ~10 8 TCID 50 / mL, The volume ratio of duck Tembusu virus CHN-YC and new duck reovirus QR-China / 2020 is 1:0.5~2.

5. The duck Tembusu virus and novel duck reovirus combined inactivated vaccine according to claim 4, characterized in that: In the two-inactivated vaccine, the content of duck Tembusu virus CHN-YC is 10 7.7 TCID 50 / mL, the content of novel duck reovirus QR-China / 2020 was 10 7.1 TCID 50 / mL, The volume ratio of duck Tembusu virus CHN-YC and the new duck reovirus QR-China / 2020 strain is 1:

1.

6. The duck Tembusu virus and novel duck reovirus combined inactivated vaccine according to claim 3, characterized in that: The inactivated vaccine also includes a pharmaceutically acceptable carrier.

7. The duck Tembusu virus and novel duck reovirus combined inactivated vaccine according to claim 6, characterized in that: The carrier is an adjuvant.

8. The duck Tembusu virus and novel duck reovirus combined inactivated vaccine according to claim 7, characterized in that: The adjuvant is any one or more of white oil, Siben-80, Tween-80 and aluminum stearate.

9. A method for preparing the duck Tembusu virus and novel duck reovirus combined inactivated vaccine according to claim 3, characterized in that: The following steps are involved: 1) Preparation of Duck Tembusu Virus CHN-YC Fluid The duck Tembusu virus CHN-YC strain was inoculated on BHK-21 cells at a dose of 0.01 to 0.1 MOI by adsorption inoculation, and the virus was collected 48 to 60 hours after inoculation. The virus titer of the virus solution was determined to be 10 -6.7 / 0.1mL; 2) Preparation of the novel duck reovirus QR-China / 2020 virus solution The novel duck reovirus QR-China / 2020 strain was inoculated on Vero cells at a dose of 0.1-0.2 MOI using a non-adsorption inoculation method, and the virus was collected 72-96 hours after inoculation. After the virus was collected, the virus titer of the virus liquid was measured, and the virus titer of the novel duck reovirus QR-China / 2020 strain was measured to be 10 -6.1 / 0.1mL; 3) Preparation of mixed virus solution The virus solution of duck Tembusu virus CHN-YC and the virus solution of novel duck reovirus QR-China / 2020 were mixed at a volume ratio of 1:0.5-2; the inactivated mixed virus solution was obtained by inactivation. 4) Preparation of the combined inactivated vaccine a. Weigh 94 parts of medical white oil, 6 parts of Siben-80 and 2 parts of aluminum stearate in parts by weight, mix thoroughly and sterilize by autoclave at 121 ° C for 15 min, cool to room temperature to obtain the oil phase and set aside; b. Weigh 96 parts by weight of the inactivated virus mixture and 4 parts of sterilized Tween-80; stir evenly as the aqueous phase; c. Weigh the oil phase and the water phase in a volume ratio of 2:1; slowly stir the oil phase in a high-speed emulsifier while slowly adding the water phase. After the addition, stir at a speed of 10,000 r / min for 5 to 15 minutes to obtain a two-inactivated vaccine. After packaging, store it at 4°C. The content of duck Tembusu virus CHN-YC in the vaccine is 10 7 ~10 8 TCID 50 / mL, the content of novel duck reovirus QR-China / 2020 was 10 7 ~10 8 TCID 50 / mL.

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