Large-scale culture method of novel duck reovirus

Abstract

According to the large-scale culture method of the novel duck reovirus, the LMH suspension cells are cultured under the serum-free condition, the influence of serum on virus production is avoided, and the production cost is reduced. The method is used for large-scale industrial production of a 10-500L bioreactor, and is high in yield, strong in stability and low in batch difference. The virus titer cultured by the LMH suspension cell provided by the invention can reach up to 109.50 TCID50 / 0.1 mL, the novel duck reovirus vaccine prepared by the LMH suspension cell is good in immune protection effect and high in vaccine safety, and a reliable technical scheme is provided for prevention and control of the novel duck reovirus disease.

Description

Technical Field

[0001] The present invention relates to the field of veterinary biological products, and in particular to a large-scale culture method of a novel duck reovirus. Background Art

[0002] Novel duck reovirus disease, caused by the novel duck reovirus (NDRV), is a major epidemic threatening my country's waterfowl industry. Clinically, the disease manifests as decreased egg production in breeder ducks, reduced hatching rates in ducklings, and poor growth and development in broiler ducks. Autopsies reveal varying degrees of enlargement, hemorrhage, and necrosis in the liver and spleen. The disease can also cause immunosuppression in infected animals, often leading to secondary or mixed infections, increased mortality, and severe economic losses.

[0003] The novel duck reovirus can be cultured in a variety of cell types, including chicken embryo fibroblasts (CEF), baby hamster kidney cells (BHK-21), duck embryo fibroblasts (DEF), or African green monkey kidney cells (Vero) for in vitro passage. However, most of these cultures rely on traditional adherent cultures, which require trypsin digestion and frequent passages. This is a cumbersome and labor-intensive process, increasing the risk of contamination. Furthermore, fetal bovine serum (FBS) must be added to the culture medium, which is expensive and subject to batch variation, affecting the stability of the virus culture and making it difficult to meet the needs of large-scale production. Currently, suspension cultures of novel duck reovirus are mostly limited to small-scale shake flask experiments. Therefore, it is urgent to establish an industrialized production process for the novel duck reovirus that can achieve stable, efficient, simple, and low-cost large-scale production. Summary of the Invention

[0004] The purpose of the present invention is to provide a large-scale culture method for a novel duck reovirus, so as to solve the problem in the prior art that the novel duck reovirus is difficult to achieve stable and efficient large-scale culture.

[0005] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0006] The invention discloses a large-scale culture method of a novel duck reovirus, wherein the culture method adopts LMH suspension cells for culture.

[0007] Furthermore, the LMH suspension cells were inoculated with the virus at a volume ratio of 0.01%.

[0008] Furthermore, the LMH suspension cell inoculation density was 2.0×10 6 cells / mL.

[0009] Furthermore, the titer of the novel duck reovirus virus solution is 10 9.38~10 9.50 TCID 50 / 0.1mL.

[0010] Furthermore, the conditions for culturing the LMH suspension cells are: temperature 37° C., dissolved oxygen 50%-60%, pH 7.0±0.2, culture speed 65-135 r / min, and culture for 72 h.

[0011] Furthermore, the LMH suspension cells are cultured in a serum-free medium.

[0012] Furthermore, the culture method is used for large-scale culture in 10L-500L bioreactors.

[0013] The invention relates to an application of the large-scale culture method of the novel duck reovirus in the preparation of a novel duck reovirus vaccine.

[0014] Compared with the prior art, the present invention has the following advantages:

[0015] The culture method provided by the present invention can be used for large-scale industrial production in 10L-500L bioreactors, and has the advantages of simple operation, high yield, strong stability and low batch-to-batch variation.

[0016] This culture method does not add serum throughout the entire process, avoiding the impact of serum on virus production and reducing production costs;

[0017] The titer of the virus cultured in the LMH suspension cells of the present invention is increased to not less than 10 9.38 TCID 50 / 0.1mL, up to 10 9.50 TCID 50 / 0.1mL;

[0018] The new duck reovirus cultured by this culture method can be used to prepare an inactivated vaccine. The vaccine has good safety and excellent immune protection effect, providing a reliable technical solution for the prevention and control of the new duck reovirus disease.

[0019] The novel duck reovirus can proliferate in a variety of cell lines, including chicken embryo fibroblasts (CEF), duck embryo fibroblasts (DEF), baby hamster kidney cells (BHK-21), and African green monkey kidney (Vero) cells. The virus exhibits significant differences in its ability to proliferate in different cell lines, exhibiting excellent proliferation properties in BHK-21 cells and causing significant cytopathic effects. Therefore, prior art has primarily used BHK-21 cells for in vitro propagation of the novel duck reovirus. Large-scale cultivation of the novel duck reovirus using LMH cells, particularly LMH suspension cells, is rare. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The accompanying drawings, which constitute part of the present invention, are provided to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are provided to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings:

[0021] Figure 1 . The liver and spleen of the experimental group of immunized ducks in the immune challenge protection experiment were examined after autopsy. A shows a normal liver after challenge, and B shows a normal spleen after challenge.

[0022] Figure 2 . The liver and spleen of the positive control group of the immune duck in the immune challenge protection experiment were examined after autopsy. A shows a normal liver after challenge, and B shows a normal spleen after challenge;

[0023] Figure 3 .These are pictures of the liver and spleen of the blank control group ducks after autopsy in the immune challenge protection experiment. A shows liver necrosis after challenge, and B shows spleen necrosis and hemorrhage after challenge. DETAILED DESCRIPTION

[0024] The following specific descriptions are exemplary and are intended to provide further explanation of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, and such modifications and replacements shall fall within the scope of protection of the present invention.

[0025] The raw materials and reagents used in the present invention are all commercially available drugs and reagents. The novel duck reovirus XT18 strain used in the examples was identified and provided by the research group of Professor Diao Youxiang of Shandong Agricultural University. It is classified and named as the novel duck reovirus N-DRV-XT18 strain and is currently deposited in the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with a deposit number of CCTCC NO: V201843 and a deposit date of July 18, 2018.

[0026] Example 1: Optimization of shake flask culture process for novel duck reovirus

[0027] 1.1. Recovery and subculture of LMH suspension cells

[0028] Remove the frozen LMH suspension cells from liquid nitrogen and thaw rapidly in a 37°C water bath. Add approximately 15-20 mL of LMH-SFM-III serum-free medium to the revived cells, centrifuge at 800 rpm for 5 minutes, discard the supernatant, and resuspend the cells in 20 mL of LMH-SFM-III serum-free medium and pipette evenly.

[0029] The cells were inoculated into 125 mL shake flasks and cultured in a shaker at 37°C, 5% CO2, and 135 rpm.

[0030] After culturing the cells for 48 h, samples were taken for counting and cell viability was calculated. 6 ~6.0×10 6 cells / mL, and when the viability was above 95%, the cell density was adjusted to 1.5×10 cells / mL using LMH-SFM-Ⅲ serum-free medium. 6 cells / mL, and cultured in separate bottles;

[0031] After three generations of continuous passages, when the cell doubling rate is stable, the cells are used for inoculation of the new duck reovirus.

[0032] 1.2 Determination of the optimal cell density for infection with the novel duck reovirus

[0033] The cell density of LMH suspension was adjusted to 1.0 × 10 6 cells / mL, 2.0×10 6 cells / mL、3.0×10 6 cells / mL, and the new duck reovirus XT18 strain (TCID 50 =10 -7.5 After the inoculation, the culture flask was placed in a shaker at 37°C, 5% CO2, and a speed of 135 r / min for 72 h. The virus solution was harvested and frozen and thawed once, and the TCID of the virus was determined. 50 The test results are shown in Table 1.

[0034] Table 1: Comparison of titer detection results of novel duck reovirus XT18 strain cultured at different inoculated cell densities

[0035]

[0036] The results showed that when the cell density was 2.0×10 6 cells / mL, and the highest titer of the new duck reovirus liquid was obtained, which could reach 10 9.38 ~10 9.50 TCID 50 / 0.1mL.

[0037] 1.3 Determination of the optimal dose of novel duck reovirus

[0038] Adjust the LMH suspension cell density to 2.0 × 10 6 cells / mL, and the new duck reovirus XT18 strain (TCID 50 =10 -7.5After the inoculation, the culture flask was placed in a shaker at 37°C, 5% CO2, and a speed of 135 r / min for 72 h. The virus solution was harvested and frozen and thawed once, and the TCID of the virus was determined. 50 The test results are shown in Table 2.

[0039] Table 2: Comparison of titer detection results of novel duck reovirus XT18 strain cultured at different inoculation doses

[0040]

[0041]

[0042] The results showed that the titer of the new duck reovirus liquid was the highest when the virus was inoculated at a volume ratio of 0.01%, which could reach 10 9.38 ~10 9.50 TCID 50 / 0.1mL.

[0043] 1.4 Determination of the optimal collection time for novel duck reovirus

[0044] Adjust the LMH suspension cell density to 2.0 × 10 6 cells / mL, and the new duck reovirus XT18 strain (TCID 50 =10 -7.5 After the inoculation, the culture flask was placed in a shaker at 37°C, 5% CO2, and a speed of 135 r / min and cultured for 120 h; samples were taken every 12 h after inoculation, and the samples were frozen and thawed once to determine the TCID of the virus. 50 The test results are shown in Table 3.

[0045] Table 3: Comparison of titer detection results of novel duck reovirus XT18 strain cultured at different virus collection times

[0046]

[0047] The results showed that the titer of the new duck reovirus liquid collected 72 hours after infection was the highest, at 10 9.38 TCID 50 / 0.1mL. 1.5. Determination of the optimal culture temperature for the novel duck reovirus

[0048] Adjust the LMH suspension cell density to 2.0 × 10 6 cells / mL, and the new duck reovirus XT18 strain (TCID 50 =10 -7.5After the inoculation, the culture flask was placed in a shaker with 5% CO2 and a rotation speed of 135 r / min for incubation. The culture temperature was set to 33°C, 35°C, 37°C, and 39°C respectively. The virus liquid was harvested 72 hours after inoculation, frozen and thawed once, and the TCID of the virus was determined. 50 The test results are shown in Table 4.

[0049] Table 4: Comparison of titer detection results of novel duck reovirus XT18 strain cultured at different temperatures

[0050]

[0051] The results showed that the titer of the new duck reovirus liquid obtained by culturing at 37℃ after infection was the highest, reaching 10 9.38 ~10 9.50 TCID 50 / 0.1mL.

[0052] 1.6. Virus titer determination method

[0053] The new duck reovirus liquid was harvested and serially diluted 10-fold with DMEM / F12 culture medium. -7 , 10 -8 , 10 -9 , 10 -10 Four dilutions were used to seed confluent monolayers of LMH adherent cells (96-well cell culture plates). Five wells of each dilution were inoculated with 0.1 mL of the virus per well. Virus-positive control wells and cell-negative control wells were also established. The cells were cultured in a 37°C, 5% CO2 incubator for 120 h. Cytopathic effects were observed daily under an inverted microscope, and viral load was calculated using the Reed-Muench method.

[0054] Example 2: Comparative experiment on culturing novel duck reovirus in different cells

[0055] Adjust the LMH suspension cell density to 2.0 × 10 6 cells / mL, and the new duck reovirus XT18 strain (TCID 50 =10 -7.5 After the inoculation, the culture flask was placed in a shaker at 37°C, 5% CO2, and a speed of 135 r / min for 72 h. When the cell density was lower than 1.0 × 10 6 cells / mL. When the cell viability dropped below 50%, the virus solution was harvested, frozen and thawed once, and the TCID of the virus was determined. 50 .

[0056] LMH adherent cells, BHK-21 adherent cells and Vero adherent cells that grew well were taken, the cell culture medium (DMEM / F12 culture medium containing 6% fetal bovine serum) was discarded, and 0.01% new duck reovirus XT18 strain (TCID 50 =10 -7.5 / 0.1mL) of virus seed cell maintenance medium (DMEM / F12 culture medium containing 1% fetal bovine serum) was placed in a 37°C, 5% CO2 environment for 72 hours. When the cells showed syncytial lesions characterized by spherical fusion and the lesion rate reached more than 90%, the virus solution was harvested, frozen and thawed once, and the TCID of the virus was determined. 50 .

[0057] The density of BHK-21 suspension cells was adjusted to 2.0 × 10 6 cells / mL, and the new duck reovirus XT18 strain (TCID 50 =10 -7.5 After the inoculation, the culture flask was placed in a shaker at 37°C, 5% CO2, and a speed of 135 r / min for 72 h. When the cell density was lower than 1.0 × 10 6 cells / mL. When the cell viability dropped below 50%, the virus solution was harvested, frozen and thawed once, and the TCID of the virus was determined. 50 The test results are shown in Table 5.

[0058] Table 5: Comparison of titer detection results of novel duck reovirus XT18 strain in different cell cultures

[0059]

[0060]

[0061] Compared with the virus titers of LMH, BHK-21, Vero adherent cells and BHK-21 suspension cells inoculated with the virus, the virus titers of LMH suspension cells inoculated with the new duck reovirus were higher, reaching 10 9.38 ~10 9.50 TCID 50 / 0.1mL.

[0062] Example 3: Large-scale cultivation of novel duck reovirus in bioreactors of different volumes

[0063] 3.1 Recovery and subculture of LMH suspension cells

[0064] Take out the frozen LMH suspension cells from liquid nitrogen and thaw them quickly in a 37°C water bath;

[0065] Add about 15-20 mL of LMH-SFM-III serum-free medium to the recovered cells, centrifuge at 800 rpm for 5 minutes, discard the supernatant, add 20 mL of LMH-SFM-III serum-free medium, resuspend the cells and pipette evenly;

[0066] The cells were inoculated into 125 mL shake flasks and cultured in a shaker at 37°C, 5% CO2, and 135 rpm.

[0067] After culturing the cells for 48 h, samples were taken for counting and cell viability was calculated. 6 ~6.0×10 6 cells / mL, and when the viability was above 95%, the cell density was adjusted to 1.5×10 cells / mL using LMH-SFM-Ⅲ serum-free medium. 6 cells / mL and culture in separate flasks.

[0068] 3.2. Scale-up culture in 10L bioreactor

[0069] The density of LMH suspension cells was adjusted to 1.5 × 10 6 cells / mL, inoculated into a 10L bioreactor with a working volume of 4L, a rotation speed of 100r / min, a dissolved oxygen content (DO) of 50%, a pH value of 7.0±0.2, and a culture temperature of 37°C. Cultured for 35-40h, the cell density was 4.0×10 6 ~4.5×10 6 cells / mL, add LMH-SFM-Ⅲ serum-free medium to adjust the cell density to 2.0×10 6 cells / mL, and the virus content (TCID 50 ) is 10 -7.5 / 0.1mL of the new duck reovirus XT18 strain, the rotation speed was 115r / min, the dissolved oxygen (DO) was 50%, the pH value was 7.0±0.2, and the culture temperature was 37℃. After 72h of culture, when the viable cell density was less than 1.0×10 6 cells / mL. When the cell viability dropped below 50%, the virus solution was harvested, frozen and thawed once, and the TCID of the virus was determined. 50 The test results are shown in Table 6.

[0070] Table 6: Virus content determination results of the novel duck reovirus XT18 strain cultured in a 10L bioreactor

[0071]

[0072] The results showed that in a 10L bioreactor, when the cell density was 2.0×10 6 cells / mL, the virus was inoculated at a volume ratio of 0.01%, and the virus solution was harvested at 10 9.38 ~10 9.50 TCID 50 / 0.1mL.

[0073] 3.3. 100L bioreactor scale-up culture

[0074] The density of LMH suspension cells was adjusted to 1.5 × 10 6 cells / mL, inoculated into a 100L bioreactor with a working volume of 40L, a rotation speed of 90r / min, a dissolved oxygen content (DO) of 50%, a pH value of 7.0±0.2, and a culture temperature of 37°C. Cultured for 35-40h, the cell density was 4.0×10 6 ~4.5×10 6 cells / mL, add LMH-SFM-Ⅲ serum-free medium to adjust the cell density to 2.0×10 6 cells / mL, and the virus content (TCID 50 ) is 10 -7.5 / 0.1mL of the new duck reovirus XT18 strain, the rotation speed was 95r / min, the dissolved oxygen (DO) was 50%, the pH value was 7.0±0.2, and the culture temperature was 37℃. After 72h of culture, when the viable cell density was less than 1.0×10 6 cells / mL. When the cell viability dropped below 50%, the virus solution was harvested, frozen and thawed once, and the TCID of the virus was determined. 50 The test results are shown in Table 7.

[0075] Table 7: Virus content determination results of the novel duck reovirus XT18 strain cultured in a 100L bioreactor

[0076]

[0077] The results showed that in a 100L bioreactor, when the cell density was 2.0×10 6 cells / mL, the virus was inoculated at a volume ratio of 0.01%, and the virus solution was harvested at 10 9.38 ~10 9.50 TCID 50 / 0.1mL.

[0078] 3.4. 500L bioreactor scale-up culture

[0079] The LMH suspension cell density was adjusted to 1.5 × 10 6 cells / mL, inoculated into a 500L bioreactor with a working volume of 200L, a rotation speed of 60r / min, a dissolved oxygen content (DO) of 50%, a pH value of 7.0±0.2, and a culture temperature of 37°C. Cultured for 35-40h, the cell density was 4.0×10 6 ~4.5×10 6 cells / mL, add LMH-SFM-Ⅲ serum-free medium to adjust the cell density to 2.0×10 6 cells / mL, and the virus content (TCID 50 ) is 10 -7.5 / 0.1mL of the new duck reovirus XT18 strain, the rotation speed was 65r / min, the dissolved oxygen (DO) was 50%, the pH value was 7.0±0.2, and the culture temperature was 37℃. After 72h of culture, when the viable cell density was less than 1.0×10 6 cells / mL. When the cell viability dropped below 50%, the virus solution was harvested, frozen and thawed once, and the TCID of the virus was determined. 50 The test results are shown in Table 8.

[0080] Table 8: Virus content determination results of the novel duck reovirus XT18 strain cultured in a 500L bioreactor

[0081]

[0082] The results showed that in a 500L bioreactor, when the cell density was 2.0×10 6 cells / mL, the virus was inoculated at a volume ratio of 0.01%, and the virus solution was harvested at 10 9.38 ~10 9.50 TCID 50 / 0.1mL.

[0083] Example 4: Preparation of a novel inactivated duck reovirus vaccine and immune protection test

[0084] The virus contents of the three batches of novel duck reovirus liquids harvested in step 3 of Example 3 were adjusted to 10 7.5 TCID 50 / 0.1mL, add 10% formaldehyde solution to it, mix thoroughly, make the final concentration of formaldehyde solution in the virus liquid to 0.2%, and inactivate at 37℃ for 24h. After testing for complete inactivation and sterility, take 96 parts of the inactivated antigen solution, add 4 parts of sterilized Tween-80, stir thoroughly until Tween-80 is completely dissolved, which is the water phase. Take 94 parts of white oil for injection, add 6 parts of Siben-80, mix thoroughly, and sterilize under high pressure to obtain the oil phase. According to the ratio of water phase: oil phase = 1:2, slowly add the water phase to the oil phase, use an emulsifier to pre-emulsify for 1 minute, then use 25000r / min for high-speed shear emulsification, emulsify for 5 minutes, and prepare 3 batches of new duck reovirus inactivated vaccine. A positive control group inactivated vaccine was set up, and the virus liquid for vaccine preparation was obtained by inoculating LMH adherent cells with the new duck reovirus seed virus. The virus content of the harvested virus liquid was adjusted to 10 7.5 TCID 50 / 0.1mL, and inactivated with formaldehyde, and the inactivated vaccine was obtained according to the same method as above.

[0085] Ten five-day-old Cherry Valley ducks were immunized subcutaneously with 1.0 mL of inactivated vaccine from each of the three experimental batches and the positive control group. Safety observations were conducted for 14 days following immunization. Survival of the test ducks was recorded, and the injection site was observed.

[0086] Safety observation experiment results: During the observation period, all the test ducks were healthy and no local or systemic adverse reactions occurred, indicating that the vaccine is safe.

[0087] Ten 5-day-old Cherry Valley ducks were immunized subcutaneously in the neck with 0.5 mL of inactivated vaccine from each of the three batches of inactivated vaccine in the experimental group and the positive control group. Ten Cherry Valley ducks of the same age were not immunized and served as the blank control group. 21 days after immunization, blood was collected from the immunized and control groups, and serum was separated to determine the neutralizing antibody titer. The new duck reovirus solution was then intravenously inoculated at 0.5 mL / bird (containing 10 6.5 TCID 50 ), clinical symptoms of the test ducks were observed after challenge and autopsied 7 days after challenge, liver and spleen lesions of the test ducks were recorded, and the challenge protection rate was calculated. The results are shown in Table 9.

[0088] Table 9: Results of neutralizing antibody titer determination and challenge protection in 5-day-old Cherry Valley ducks after vaccination

[0089]

[0090] The results of the immune challenge protection experiment showed that the liver and spleen of the experimental group and the positive control group were normal after autopsy after the immune challenge (see Figure 1 and Figure 2), the protection rate of the challenge was 100%; the autopsy of the blank control group ducks after challenge showed typical lesions of liver necrosis and spleen necrosis and hemorrhage (see Figure 3 ), with an incidence rate of 100%.

[0091] The results showed that the inactivated vaccine prepared from the novel duck reovirus liquid obtained in step 3 of Example 3 had a good immune protection effect.

[0092] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A large-scale culture method for a novel duck reovirus, characterized in that: The culture method is to culture LMH suspension cells in vitro.

2. The large-scale culture method of the novel duck reovirus according to claim 1, characterized in that: The LMH suspension cells were inoculated with the virus at an inoculation dose of 0.01% by volume.

3. The large-scale culture method of the novel duck reovirus according to claim 1, characterized in that: The inoculation density of the LMH suspension cells was 2.0×10 6 cells / mL.

4. The large-scale culture method of the novel duck reovirus according to claim 1, characterized in that: The titer of the novel duck reovirus fluid is 10 9.38 ~10 9.50 TCID 50 / 0.1mL.

5. The large-scale culture method of the novel duck reovirus according to claim 1, characterized in that: The conditions for culturing the LMH suspension cells are: temperature 37° C., dissolved oxygen content 50%-60%, pH value 7.0±0.2, culture speed 65-135 r / min, and culture for 72 hours.

6. The large-scale culture method of the novel duck reovirus according to claim 1, characterized in that: The LMH suspension cells were cultured using serum-free medium.

7. The large-scale culture method of the novel duck reovirus according to claim 1, characterized in that: The culture method is used for large-scale culture in 10L-500L bioreactors.

8. Use of the large-scale culture method of the novel duck reovirus according to claim 1 in preparing a novel duck reovirus vaccine.

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