A novel duck reovirus NDRV-HZ strain suspension culture serum-free medium SF-1 and a preparation method thereof

Abstract

The application belongs to the technical field of veterinary biological products, and discloses a novel duck reovirus NDRV-HZ strain suspension culture serum-free medium SF-1 and a preparation method thereof. The application discloses a culture medium additive, and the culture medium additive comprises Zn 2+ , L-arginine hydrochloride and pyridoxine hydrochloride. The serum-free medium SF-1 for suspension culture provided by the application significantly improves the replication titer of the NDRV-HZ virus, and the TCID 50 / mL under the conventional culture condition is 10 6.0 , which is improved to 10 8.50 , with an increase of 316 times. The application determines key parameters such as inoculation MOI=0.05, culture temperature 37 DEG C and virus collection time window 48-54h, and significantly improves the virus preparation efficiency. Further, the application develops a high-efficiency inactivated vaccine, and provides an innovative solution for the prevention and control of duck reovirus disease.

Description

Technical Field

[0001] This invention belongs to the field of veterinary biological products technology, specifically relating to a serum-free culture medium SF-1 for suspension culture of a novel duck reovirus NDRV-HZ strain and its preparation method. Background Technology

[0002] Duck reovirus disease is an important infectious disease of waterfowl caused by members of the genus Orthoreovirus in the family Reoviridae. In recent years, the prevalence of novel duck reovirus (NDRV) in the duck farming industry has become increasingly serious. This virus mainly infects ducklings aged 1-4 weeks, causing hemorrhage and necrosis of internal organs such as the liver and spleen, with a mortality rate of 10%-30%, resulting in huge economic losses to the duck farming industry.

[0003] Currently, the development of NDRV vaccines mainly faces the following technical bottlenecks: (1) Culture medium limitations: Traditional virus culture mostly adopts adherent cell culture, which has problems such as high labor intensity, high production cost and low virus yield. Although suspension culture technology can achieve large-scale production, the conventional SF basic suspension medium has limited support for NDRV-HZ virus replication, and the virus titer can usually only be maintained at TCID. 50 / mL=106.0, which is difficult to meet the needs of industrial production of vaccines; (2) The culture process is not mature: the key process parameters such as the optimal multiple of infection (MOI), culture temperature and virus collection time of NDRV-HZ in suspension cells have not been systematically optimized, resulting in low virus replication efficiency and a culture cycle of 72-80h; (3) Insufficient immunogenicity: the inactivated vaccine prepared by low titer virus solution has poor immunogenicity and requires increased immunization dose or increased number of immunizations to achieve the ideal protective effect.

[0004] Therefore, developing a modified culture medium that can significantly improve the replication titer of NDRV-HZ virus and establishing a corresponding suspension culture process is of great practical significance for the industrial production of a novel duck reovirus inactivated vaccine. Summary of the Invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art.

[0006] The first aspect of the present invention is to provide a culture medium additive.

[0007] A second aspect of the present invention is to provide a serum-free culture medium.

[0008] A third aspect of the present invention aims to provide the use of the culture medium additive of the first aspect of the present invention and the serum-free culture medium of the second aspect of the present invention in the preparation of products for treating and / or treating diseases caused by duck reovirus.

[0009] The fourth aspect of this invention is to provide a method for promoting the proliferation of duck reovirus.

[0010] The fifth aspect of this invention is to provide a vaccine.

[0011] The sixth aspect of this invention is to provide a method for preparing the vaccine according to the fifth aspect of this invention.

[0012] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A first aspect of the present invention provides a culture medium additive comprising Zn 2+ L-arginine hydrochloride and pyridoxine hydrochloride.

[0013] In some embodiments of the present invention, the Zn 2+ The addition amount is 15-45 μM.

[0014] In some embodiments of the present invention, the Zn 2+ The addition amount is 20-40 μM; for example, it can be any value of 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 or 40 μM or any range between two.

[0015] In some embodiments of the present invention, the Zn 2+ It includes at least one of ZnSO4, ZnCl2 and zinc citrate.

[0016] The Zn 2+ Zn is an essential cofactor of NDRV-HZ viral RNA-dependent RNA polymerase (RdRp), which promotes viral genome replication. Meanwhile, Zn... 2+ It can stabilize the viral capsid protein structure and improve the assembly efficiency of viral particles. Appropriate amounts of Zn... 2+ It can also enhance the antioxidant capacity of host cells and maintain the metabolic activity of cells during viral infection.

[0017] In some embodiments of the present invention, the amount of L-arginine hydrochloride added is 100-150 mg / L.

[0018] In some embodiments of the present invention, the amount of L-arginine hydrochloride added is 115-140 mg / L; for example, it can be any value or a range between 115, 118, 121, 124, 127, 130, 133, 136, 139 or 140 mg / L.

[0019] L-arginine is an important nitrogen precursor for the synthesis of viral structural and non-structural proteins. Adding L-arginine hydrochloride significantly increases intracellular arginine levels, promoting efficient viral protein synthesis. Simultaneously, L-arginine regulates the host cell's immune response through the nitric oxide (NO) signaling pathway, creating a favorable intracellular environment for viral replication.

[0020] In some embodiments of the present invention, the amount of pyridoxine hydrochloride added is 0.5-2.5 mg / L.

[0021] In some embodiments of the present invention, the amount of pyridoxine hydrochloride added is 1-2 mg / L; for example, it can be any value or a range between 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 mg / L.

[0022] Pyridoxine hydrochloride, as a precursor of pyridoxal phosphate (PLP), is a coenzyme for amino acid transaminases and decarboxylases. Adding pyridoxine hydrochloride promotes amino acid metabolism in host cells, accelerating the supply of amino acids required for viral protein synthesis. Furthermore, pyridoxine participates in glycogen and lipid metabolism, providing ample energy support for viral replication.

[0023] A second aspect of the present invention provides a serum-free culture medium comprising a basal culture medium and a culture medium additive of the first aspect of the present invention.

[0024] In some embodiments of the present invention, the basal culture medium includes at least one of DMEM medium, MEM medium, DMEM / F12 medium, F10 medium, F12 medium and IMDM medium.

[0025] A third aspect of the invention provides the use of the culture medium additive of the first aspect of the invention or the serum-free culture medium of the second aspect of the invention in the preparation of products for treating and / or treating diseases caused by duck reovirus.

[0026] In some embodiments of the present invention, the product includes reagents, drugs, and / or egg yolk antibodies.

[0027] In some embodiments of the present invention, the reagent includes diagnostic reagents.

[0028] In some embodiments of the present invention, the diagnostic reagent includes an antigen for diagnosing duck reovirus, the antigen being an inactivated duck reovirus of the first aspect of the present invention.

[0029] In some embodiments of the present invention, the drug includes a vaccine.

[0030] In some embodiments of the present invention, the vaccine includes an inactivated vaccine.

[0031] In some embodiments of the invention, the disease includes enlarged and necrotic livers in ducklings caused by duck reovirus infection.

[0032] In some embodiments of the present invention, the duck reovirus is duck reovirus NDRV-HZ (DuckReovirus), which was deposited at the China Center for Type Culture Collection on April 21, 2026, with accession number CCTCC NO:V202645.

[0033] The strain was isolated from ducklings with enlarged and necrotic livers and was identified as duck reovirus by gene sequencing and RT-PCR.

[0034] A fourth aspect of the present invention provides a method for promoting the proliferation of duck reovirus, the method comprising the following steps: Cells are cultured using the serum-free culture medium of the second aspect of the present invention to obtain cells in suspension culture; The duck reovirus was inoculated into the suspended cultured cells, adsorbed, and amplified.

[0035] In some embodiments of the present invention, the duck reovirus is duck reovirus NDRV-HZ, which was deposited at the China Center for Type Culture Collection on April 21, 2026, with accession number CCTCC NO:V202645.

[0036] In some embodiments of the present invention, the cell is a mammalian cell.

[0037] In some embodiments of the present invention, the mammalian cells are chicken liver cancer cells.

[0038] In some embodiments of the present invention, the MOI of the duck reovirus inoculation is 0.001-0.1.

[0039] In some embodiments of the present invention, the MOI of the duck reovirus inoculation is 0.05-0.1; for example, it can be any value of 0.05, 0.06, 0.07, 0.08, 0.09 or 0.1 or a range between any two.

[0040] In some embodiments of the present invention, the adsorption temperature of the duck reovirus is 30-38°C.

[0041] In some embodiments of the present invention, the adsorption temperature of the duck reovirus is 33-38°C; for example, it can be any value or a range between any two of 33, 33.5, 34, 34.5, 35, 35.5, 36, 36.5, 37, 37.5 or 38°C.

[0042] In some embodiments of the present invention, the adsorption time is 36-66 hours.

[0043] In some embodiments of the present invention, the adsorption time is 42-60 h; for example, it can be any value of 42, 45, 48, 51, 54, 57 or 60 h or a range between any two.

[0044] In some embodiments of the present invention, the adsorption time is 48-54 h; for example, it can be any value of 48, 49, 50, 51, 52, 53 or 54 h or a range between any two.

[0045] A fifth aspect of the present invention provides a vaccine, the active ingredient of which comprises an inactivated duck reovirus.

[0046] In some embodiments of the present invention, the duck reovirus is duck reovirus NDRV-HZ, which was deposited at the China Center for Type Culture Collection on April 21, 2026, with accession number CCTCC NO:V202645.

[0047] In some embodiments of the present invention, the vaccine further includes pharmaceutically acceptable excipients.

[0048] In some embodiments of the present invention, the excipients include at least one of a carrier, a protective agent, an adjuvant, an excipient, or a diluent.

[0049] In some embodiments of the present invention, the excipients include Tween, white oil, and Span.

[0050] In some embodiments of the present invention, the immunization methods of the vaccine include nasal drops, injection, oral administration, soaking, or eye drops.

[0051] In some embodiments of the present invention, the vaccine is administered by injection.

[0052] In some embodiments of the present invention, the vaccine is administered via intramuscular injection.

[0053] A sixth aspect of the present invention provides a method for preparing the vaccine of the fifth aspect of the present invention, comprising the following steps: Duck reovirus was cultured and multiplied in cells to obtain viral fluid; or duck reovirus was inoculated into specific pathogen-free duck embryos, incubated, and allantoic fluid from surviving embryos was collected to obtain viral fluid. Inactivation, resulting in an inactivated vaccine.

[0054] In some embodiments of the present invention, the duck reovirus is duck reovirus NDRV-HZ, which was deposited at the China Center for Type Culture Collection on April 21, 2026, with accession number CCTCC NO:V202645.

[0055] In some embodiments of the present invention, the culture and proliferation includes the following steps: inoculating duck reovirus into cells in suspension culture, and adding Zn to the culture system. 2+ L-arginine hydrochloride and pyridoxine hydrochloride are adsorbed and amplified in culture of the duck reovirus.

[0056] In some embodiments of the present invention, the Zn 2+ The addition amount is 15-45 μM.

[0057] In some embodiments of the present invention, the Zn 2+ The addition amount is 20-40 μM; for example, it can be any value of 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 or 40 μM or any range between two.

[0058] In some embodiments of the present invention, the amount of L-arginine hydrochloride added is 100-150 mg / L.

[0059] In some embodiments of the present invention, the amount of L-arginine hydrochloride added is 115-140 mg / L; for example, it can be any value or a range between 115, 118, 121, 124, 127, 130, 133, 136, 139 or 140 mg / L.

[0060] In some embodiments of the present invention, the amount of pyridoxine hydrochloride added is 0.5-2.5 mg / L.

[0061] In some embodiments of the present invention, the amount of pyridoxine hydrochloride added is 1-2 mg / L; for example, it can be any value or a range between 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 mg / L.

[0062] In some embodiments of the present invention, the MOI of the duck reovirus inoculation is 0.001-0.1.

[0063] In some embodiments of the present invention, the MOI of the duck reovirus inoculation is 0.05-0.1; for example, it can be any value of 0.05, 0.06, 0.07, 0.08, 0.09 or 0.1 or a range between any two.

[0064] In some embodiments of the present invention, the adsorption temperature of the duck reovirus is 30-38°C.

[0065] In some embodiments of the present invention, the adsorption temperature of the duck reovirus is 33-38°C; for example, it can be any value or a range between any two of 33, 33.5, 34, 34.5, 35, 35.5, 36, 36.5, 37, 37.5 or 38°C.

[0066] In some embodiments of the present invention, the adsorption time is 36-66 hours.

[0067] In some embodiments of the present invention, the adsorption time is 42-60 h; for example, it can be any value of 42, 45, 48, 51, 54, 57 or 60 h or a range between any two.

[0068] In some embodiments of the present invention, the adsorption time is 48-54 h; for example, it can be any value of 48, 49, 50, 51, 52, 53 or 54 h or a range between any two.

[0069] In some embodiments of the present invention, the inactivation includes adding formaldehyde to the virus solution and inactivating it at 35-38°C for 20-30 hours.

[0070] In some embodiments of the present invention, the final concentration of formaldehyde is 0.1%-0.3%.

[0071] The beneficial effects of this invention are: Compared with the prior art, the present invention has the following significant advantages: This invention involves adding Zn to a basic SF suspension culture medium. 2+ An improved culture medium, SF-1, was constructed using three functional components: L-arginine hydrochloride and pyridoxine hydrochloride. In this medium, the replication titer of NDRV-HZ virus increased from TCID under conventional culture conditions. 50 / mL=10 6.0 Upgrade to TCID 50 / mL=10 8.50 The increase was 316 times, with a significant increase in viral titer. This substantial increase in viral titer means a significant increase in the amount of vaccine that can be prepared per unit volume of viral fluid; conventional inactivated vaccines require 10 [units of viral fluid] per dose. 5.0 TCID 50Viral antigen calculations show that the viral antigen content harvested from the improved culture medium can meet more than 300 times the requirements of conventional culture media, thereby significantly reducing investment in culture equipment, labor costs, and energy consumption, reducing production costs, and laying a solid foundation for the large-scale production of vaccines.

[0072] This invention provides the first systematic explanation of Zn 2+ The synergistic promoting effect of L-arginine hydrochloride and pyridoxine hydrochloride on NDRV-HZ virus replication. Zn 2+ The three components, L-arginine hydrochloride and pyridoxine hydrochloride, exhibit synergistic effects with a clear mechanism of action. Zn 2+ By activating viral RNA polymerase and stabilizing capsid protein structure, the virus promotes genome replication and viral assembly; L-arginine hydrochloride provides sufficient nitrogen for viral protein synthesis and optimizes the intracellular environment through the NO signaling pathway; pyridoxine hydrochloride provides raw materials and energy for viral replication by promoting amino acid metabolism. These three components synergistically support efficient viral replication from different metabolic stages, forming a complete nutritional support system. The modified SF-1 culture medium provided by this invention only requires the addition of three common biochemical reagents to the basic SF medium. The medium formulation is simple, the raw materials are readily available, and the cost is controllable. The raw materials are widely available and inexpensive, eliminating the need for imported special culture medium components or expensive growth factors, making it suitable for the cost control needs of domestic vaccine manufacturers and possessing good prospects for industrial application.

[0073] This invention isolated a duck reovirus NDRV-HZ strain with excellent immunogenicity, constructed a serum-free suspension culture medium SF-1, and determined key parameters such as the optimal inoculation MOI of 0.05, culture temperature of 37℃, and virus harvesting time window of 48-54 h, significantly improving the efficiency of virus preparation. Using the modified SF-1 medium, the optimal virus harvesting time for NDRV-HZ was shortened from 72-80 h in conventional processes to 48-54 h, reducing the culture cycle by approximately 33%. In continuous bioreactor production, shortening the culture cycle directly increases equipment turnover and annual production capacity. Based on annual batch production, under the same production capacity conditions, the number of bioreactors can be reduced or the culture volume can be decreased, further reducing fixed asset investment and operating costs. The NDRV-HZ virus culture process provided by this invention, through optimization and determination of key parameters such as the optimal MOI (0.05), culture temperature (37℃), and virus harvesting time window (48-54 h), significantly improves the efficiency of virus preparation and is easily applicable to vaccine manufacturers.

[0074] This invention provides an inactivated vaccine prepared based on high-titer viral fluid, offering an innovative solution for the prevention and control of duck reovirus disease. The vaccine exhibits excellent immunogenicity, inducing high-titer HI antibodies (log25.8) within 3 weeks post-immunization, reaching a peak at 5 weeks (log28.5), and achieving a 100% protection rate against challenge, significantly superior to commercially available conventional vaccines (85% protection rate). Furthermore, the vaccine demonstrates good safety, with no adverse effects on the growth and development of ducklings. Attached Figure Description

[0075] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 This is an RT-PCR identification diagram of NDRV-HZ. In the diagram, M represents the DNA marker, 1-3 represent clinical cases, 4 represents the positive control, and 5 represents the negative control.

[0076] Figure 2 This image shows the RT-PCR identification of other duck viruses. In the image, M represents a DNA marker, 1 represents a novel duck reovirus, 2 represents a duck hepatitis virus, 3 represents a duck Tembusu virus, 4 represents a duck parvovirus, 5 represents a duck adenovirus, and 6 represents a duck circovirus.

[0077] Figure 3 For TCID 50 Image of CPE detected. In the image, the left side shows normal adherent LMH-HS cells, and the right side shows LMH-HS cells 7 days after viral inoculation. The scale bar is 20 μm.

[0078] Figure 4 Image showing the lesions of suspended cells in chicken liver cancer caused by a novel duck reovirus.

[0079] Figure 5 The results are from a response surface methodology experiment.

[0080] Figure 6 This is a sterility test image of a novel duck reovirus after inactivation.

[0081] Figure 7 This diagram illustrates the safety verification of the novel duck reovirus after inactivation.

[0082] Figure 8 This is a diagram illustrating the immunization effect of a novel duck reovirus inactivated vaccine. Detailed Implementation

[0083] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0084] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0085] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0086] Example 1: Isolation and Identification of a Novel Duck Reovirus NDRV-HZ Strain 1. Separation Liver tissue from ducklings with typical hepatic enlargement and necrosis was collected from a duck farm in Shandong Province. Five volumes of PBS solution were added, and the tissue blocks were ground. PBS containing penicillin (100 U / mL) and streptomycin (100 μg / mL) was added to prepare a 10% v / v suspension. The suspension was freeze-thawed three times at -80℃, centrifuged at 3000 rpm for 20 min, and the supernatant was filtered through a 0.22 μm filter. The supernatant was then inoculated into the allantoic cavity of 9-day-old specific pathogen-free duck embryos at a rate of 0.2 mL / embryo and incubated at 37℃ for 72 h. Allantoic fluid from embryos that died within 96 h and from surviving embryos was harvested and stored at -80℃.

[0087] 2. RT-PCR identification Liver, spleen, and other relevant tissues from dead duck embryos in clinical cases were sampled for RT-PCR testing. RT-PCR testing was performed simultaneously with a positive control (a known NDRV standard strain) and a negative control (healthy duck embryo tissue). Results showed that the dead duck embryo tissue samples successfully amplified a specific band of the expected size of 976 bp (see [link to relevant documentation]). Figure 1 The negative control showed no band, while the positive control showed the expected band, indicating that the isolated virus strain could effectively proliferate in duck embryos.

[0088] The harvested viral fluid was subjected to RT-PCR to detect duck reovirus, with various waterfowl viruses (duck hepatitis virus, duck Tembusu virus, duck parvovirus, duck adenovirus, and duck circovirus) used as control groups. The RT-PCR reaction system (Sewell Biotechnology Co., Ltd. One Step RT-PCR kit) consisted of: 12.5 μL of 2×One Step RT-PCR Buffer, 1 μL of One Step RT-PCR Enzyme Mix, 1 μL of upstream primer NDRV-F (10 μmol / L), 1 μL of downstream primer NDRV-R (10 μmol / L), 2 μL of template RNA, and RNase-free ddH2O to a final volume of 25 μL. The reaction program was: reverse transcription at 50℃ for 30 min; pre-denaturation at 95℃ for 5 min; denaturation at 95℃ for 30 s, annealing at 55℃ for 30 s, extension at 72℃ for 1 min, for a total of 35 cycles; final extension at 72℃ for 10 min. The nucleotide sequences of the primers used were NDRV-F: 5'-ATGAGTTCGCGCAAAGTGGCTAGACG-3' (SEQ ID NO:1); NDRV-R: 5'-CCCACATGTCAGCCCATTCAGAAG-3' (SEQ ID NO:2), with an amplification size of 976 bp. The primers were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0089] Upon identification, the isolated strain (designated NDRV-HZ) only amplified the target fragment corresponding to the novel duck reovirus. Figure 2 ), which matches the expected segment size ( Figure 1 No other viruses were detected.

[0090] Example 2: Optimization of Conventional Culture Process 1. MOI Optimization Frozen chicken liver cancer suspension cells (LMH-HS) were rapidly thawed in a 37°C water bath at a concentration of 1.0 × 10⁻⁶. 6 Cells were seeded at a density of 1 / mL in basal serum-free medium (SF) and cultured in suspension at 37°C in a 5% CO2 incubator. Cells were sampled and counted every 24 hours to monitor cell growth status.

[0091] The results showed that LMH-HS cells grew well in the modified culture medium, with cell density increasing from 1.0 × 10⁻⁶ cells / day within 72 hours. 6 Cells / mL proliferated to 7.5-8.0 × 10⁻⁶ 6 Cells / mL, cell viability maintained above 95%.

[0092] In serum-free basal medium (SF), chicken hepatocellular carcinoma suspension cells (LMH-HS cells, sourced from Guangdong Huasheng Biotechnology Co., Ltd.) were inoculated with NDRV-HZ at MOIs of 0.001, 0.005, 0.01, 0.05, 0.1, and 0.5, respectively. Viral titers were measured after incubation at 37°C for 12 h, 36 h, and 72 h. The harvested virus solution was subjected to three freeze-thaw cycles for further viral titer testing. The virus from these three freeze-thaw cycles was then serially diluted 10-fold, with the dilution range being 10-10. -1 -10 -8 The virus was seeded at a concentration of 100 μL per dilution into 96-well plates containing LMH-HS adherent cells (LMH-HS adherent cell density was 0.3 × 10⁻⁶). 6 Cells / ml were cultured for 24 hours for virus titer detection. A negative control group was set up: 100 µL of virus-free SF medium was added to observe the normal state of the cells. Cells were cultured at 37°C and 5% CO2 for 7 days. The CPE (cytopathic effect) of the cells was observed under an inverted microscope. The number of wells with CPE (positive wells) and the number of wells without CPE (negative wells) were recorded at each dilution. TCID was calculated using the Reed-Mucnch method. 50 The calculation formula is: lgTCID 50 =lgD 高 + Distance ratio × lgd. Where: Distance ratio = (P 高 -50%) / (P 高 -P 低 P 高 A cumulative positivity rate exceeding 50%; P 低 A cumulative positivity rate of less than 50%; D 高 The highest dilution with a positivity rate above 50%; d is the dilution factor (usually 10).

[0093] TCID 50 The CPE map (MOI=0.01) during detection is as follows: Figure 3 As shown, TCID 50 The results are shown in Table 1. Table 1 shows that the highest viral titer, reaching TCID, was observed when the MOI was between 0.01 and 0.05. 50 / mL=10 6.0 When the MOI is too high (≥0.1), the virus causes premature cell death, which in turn affects the virus's ability to replicate and leads to a decrease in titer. Therefore, the optimal MOI was determined to be 0.05.

[0094] Table 1. Effects of different MOIs on NDRV-HZ viral titers

[0095] 2. Optimization of culture temperature Chicken hepatoma suspension cells (LMH-HS) were cultured in basal SF medium at 33℃, 35℃, 37℃, and 39℃ and inoculated with NDRV-HZ (MOI=0.05). Viral titers were measured after 72 hours. The harvested virus solution was repeatedly frozen and thawed three times before further viral titer detection. The virus from the three freeze-thaw cycles was then serially diluted 10-fold, with the dilution range being 10-10. -1 -10 -8 The virus was seeded at a concentration of 100 μL per dilution into 96-well plates containing LMH-HS adherent cells (LMH-HS adherent cell density was 0.3 × 10⁻⁶). 6 Cells were cultured at 100 µL / ml for 24 h for virus titer detection. A negative control group was also set up: 100 µL of virus-free SF medium was added to observe the normal state of the cells. Cells were cultured at 37°C and 5% CO2. Cell growth was observed under an inverted microscope, and the time of CPE appearance was recorded. TCID was calculated using the Reed-Mucnch method. 50 .

[0096] Table 2 shows that 37℃ is the optimal temperature for NDRV-HZ virus replication, at which the viral titer is highest (TCID). 50 / mL=10 6.0 Furthermore, the timing of CPE's appearance is opportune, which is conducive to the full replication of the virus.

[0097] Table 2 Effect of different culture temperatures on NDRV-HZ virus titers

[0098] 3. Virus replication curve In basal SF medium, chicken liver cancer suspension cells LMH-HS were cultured at 37°C and inoculated with NDRV-HZ (MOI=0.05). Viral titers were measured every 6 hours, and viral proliferation curves were plotted.

[0099] As shown in Table 3, the viral titer of NDRV-HZ in basal SF medium reached its peak (TCID50) at 48-54 h. 50 / mL=10 6.0 Subsequently, due to the massive death of cells, the viral titer gradually decreased.

[0100] Table 3. NDRV-HZ virus proliferation curves (basal SF medium)

[0101] 4. Optimized poison collection time Based on the aforementioned viral replication curve, viral fluid was harvested at 48h, 54h, 60h, 66h, 72h, and 80h, respectively. The effects of different harvest times on viral titer were compared, cell debris content was observed, and a comprehensive score was calculated. The comprehensive score was based on: viral titer score (maximum 50 points) + cell debris content score (maximum 50 points), for a total score of 100 points.

[0102] As shown in Table 4, the optimal time for virus collection in basal SF medium is 48-54 hours, at which time the virus titer is the highest.

[0103] Table 4. Effects of different harvest times on NDRV-HZ virus yield

[0104] 5. Optimization of modified culture medium SF-1 5.1 Response Surface Design Use 2 3 The optimization was carried out using a partial implementation of factor design (three factors, two levels) with Zn. 2+ The concentrations (A), L-arginine hydrochloride concentration (B), and pyridoxine hydrochloride concentration (C) were used as independent variables, and the viral titer (Y, log) was used as the variable. 10 TCID 50 Using the response value ( / mL), a three-factor, two-level optimization experiment was conducted. Each factor was set to two levels, "present / absent" (0 for no addition, and the optimal concentration for addition). A total of 7 experiments were designed (1 blank control group + 3 single-factor groups + 3 two-factor combinations) to evaluate the effects of each factor individually and synergistically on NDRV-HZ virus replication.

[0105] Response surface methodology results are as follows Figure 4 As shown. Figure 4 (a) shows Zn 2+ The interaction response surface of ZnSO4 (purchased from Sinopharm Chemical Reagent Co., Ltd., catalog number: 10024018) with L-arginine hydrochloride (purchased from MacKlin, CAS No.: 15595-35-4) under the condition of fixed pyridoxine hydrochloride (Guangdong Mingtong Biotechnology Co., Ltd., CAS No.: 58-56-0) 1.5 mg / L is shown to be in the presence of ZnSO4. 2+ The viral titer reached its peak at a concentration of approximately 30 μM and an L-arginine hydrochloride concentration of approximately 126.64 mg / L. Figure 4 (b) shows Zn 2+ The interaction between pyridoxine hydrochloride and L-arginine hydrochloride at a fixed concentration of 126.64 mg / L. Figure 4 (c) shows the effect of L-arginine hydrochloride and pyridoxine hydrochloride on Zn immobilization. 2+ Interaction under 30 μM conditions Figure 4The middle (d) diagram is a three-dimensional response surface plot, which intuitively shows the synergistic effect of the three factors on the viral titer.

[0106] 5.2 Improved Culture Medium Component Optimization Based on response surface methodology optimization, the effects of three functional components on NDRV-HZ virus titers were further verified. ZnSO4 (providing Zn) was added to basal SF medium at a final concentration of 30 μM. 2+ 126.64 mg / L of L-arginine hydrochloride and 1.5 mg / L of pyridoxine hydrochloride were fully dissolved, filtered through a 0.22 μm filter membrane for sterilization, and stored at 4°C for later use. The concentrations of each single-factor component were determined based on the response surface methodology results (Zn). 2+ : 30uM; L-arginine hydrochloride: 126.64mg / L; pyridoxine hydrochloride: 1.5mg / L).

[0107] Table 5 shows that the three functional components (Zn) 2+ The addition of Zn(II) alone (such as L-arginine hydrochloride and pyridoxine hydrochloride) can increase viral titers to varying degrees. 2+ The most significant improvement was observed in the viral load. When all three components were used in combination, a significant synergistic effect was observed, with the viral titer decreasing from the TCID values ​​in the basal medium. 50 / mL=10 6.0 Upgrade to TCID 50 / mL=10 8.5 This represents an increase of approximately 316 times, and the time for virus collection has been shortened from 72-80 hours to 66-73 hours. (Image of NDRV-HZ-infected LMH-HS suspension cell lesions) Figure 5 ).

[0108] Table 5. Effects of modified SF-1 components in the culture medium on NDRV-HZ virus titers.

[0109] Example 3: Preparation of a novel duck enterovirus inactivated vaccine A method for preparing a novel inactivated duck enterovirus vaccine includes the following steps: (1) Viral proliferation Frozen chicken liver cancer suspension cells (LMH-HS) were rapidly thawed in a 37°C water bath at a concentration of 1.0 × 10⁻⁶. 6 Inoculated at a density of Cells / mL in modified SF-1 medium (SF-1 is SF basal medium supplemented with Zn). 2+LMH-HS suspension cells (30 μM; L-arginine hydrochloride: 126.64 mg / L, prepared by incubation) were placed in a 37°C, 5% CO2 incubator for suspension culture. Cell growth was monitored by sampling and counting every 24 hours. Results showed that LMH-HS suspension cells grew well in the modified medium, with cell density increasing from 1.0 × 10⁻⁶ cells / mL within 72 hours. 6 Cells / mL proliferated to 7.5-8.0 × 10⁻⁶ 6 Cells / mL, cell viability maintained above 95%.

[0110] In SF-1 medium, chicken liver cancer suspension cells LMH-HS were cultured at 37°C and inoculated with NDRV-HZ (MOI=0.05). The virus was harvested after 48-54 hours of culture.

[0111] (2) Inactivation of the virus The harvested NDRV-HZ virus solution was added to formaldehyde (purchased from Merck, catalog number: F8775-500ML) at a volume ratio of 1:1000, and inactivated by magnetic stirring at 37℃ for 24 hours. Samples were then taken for testing.

[0112] Sterility test: The inactivated virus solution was inoculated into PDA, TSA and LB medium and incubated at 37°C for 7 days.

[0113] Aseptic test results are as follows Figure 6 As shown, no microorganisms grew on PDA, TSA, and LB media.

[0114] Safety verification of inactivated virus: Twenty healthy duck embryos aged 9-11 days were randomly divided into two groups of 10 each. The first group was the experimental group, which was injected with the inactivated virus solution (TCID) prepared in Example 3. 50 / 0.1mL=10 8.5 The first group was injected with 0.2 mL of PBS solution, and the second group was the control group. The embryos were incubated at 37°C for 72 hours and their survival was observed. If the duck embryos were healthy, they were blindly passaged for one generation. If the duck embryos were still healthy, they were considered to be completely inactivated, indicating that the prepared inactivated virus solution had good safety.

[0115] The results are as follows Figure 7 As shown, the prepared inactivated vaccine has good safety.

[0116] Example 4: Safety verification of a novel duck enterovirus inactivated vaccine The novel duck reovirus inactivated vaccine prepared in Example 3 was used to immunize 30-day-old ducklings (n=20) at a dose of 1.0 mL / bird, with a 14-day interval between the second immunization. A control group (n=20) was also included. Blood samples were collected at 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks and 6 weeks after immunization. Serum was separated and the neutralizing antibody titer of the novel duck reovirus in the serum was determined. The detection method was as follows (Liu Zhe, Luo Liezhu, Zhou Xinrui, et al. Preparation and immunization study of novel duck reovirus I3-σB / σC protein vaccine [J]. Chinese Journal of Veterinary Science, 2025, 55(11):1445-1452. DOI:10.16656 / j.issn.1673-4696.2025.0201.).

[0117] Test results as follows Figure 8 As shown, the novel duck reovirus inactivated vaccine prepared in Example 3 can produce high antibody titers. It can induce high-titer HI antibodies (log25.8) 3 weeks after immunization and reach a peak (log28.5) at 5 weeks. The challenge protection rate is 100%, which is significantly better than the commercially available conventional vaccine (protection rate of 85%), and has a certain preventive effect against the virus.

[0118] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, unless otherwise specified, the embodiments of the present invention and the features thereof can be combined with each other.

Claims

1. A culture medium additive, characterized in that, The culture medium additive includes Zn. 2+ L-arginine hydrochloride and pyridoxine hydrochloride.

2. The culture medium additive according to claim 1, characterized in that, The Zn 2+ The addition amount is 15-45 μM; Preferably, the amount of L-arginine hydrochloride added is 100-150 mg / L; Preferably, the amount of pyridoxine hydrochloride added is 0.5-2.5 mg / L.

3. A serum-free culture medium, characterized in that, The serum-free culture medium includes a basal culture medium and the culture medium additives as described in claim 1 or 2; Preferably, the basal culture medium includes at least one of DMEM medium, MEM medium, DMEM / F12 medium, F10 medium, F12 medium and IMDM medium.

4. The use of the culture medium additive of claim 1 or 2 or the serum-free culture medium of claim 3 in the preparation of products for treating and / or treating diseases caused by duck reovirus.

5. The application according to claim 4, characterized in that, The products include reagents, drugs, and / or egg yolk antibodies; Preferably, the reagent includes diagnostic reagents; Preferably, the drug includes a vaccine; Preferably, the vaccine comprises an inactivated vaccine.

6. The application according to claim 4, characterized in that, The duck reovirus mentioned is duck reovirus NDRV-HZ, which was deposited at the China Center for Type Culture Collection on April 21, 2026, with accession number CCTCC NO:V202645.

7. A method for promoting the proliferation of duck reovirus, characterized in that, The method includes the following steps: Cells were cultured in the serum-free culture medium according to claim 3 to obtain cells in suspension culture. The duck reovirus was inoculated into the suspended cultured cells, adsorbed, and amplified. Preferably, the duck reovirus is the duck reovirus NDRV-HZ as described in claim 6; Preferably, the MOI of the duck reovirus inoculation is 0.001-0.1; Preferably, the adsorption temperature of the duck reovirus is 30-38℃; Preferably, the adsorption time is 36-66 hours.

8. A vaccine, characterized in that, The active ingredient of the vaccine includes the inactivated duck reovirus as described in claim 6.

9. The vaccine according to claim 8, characterized in that, The vaccine also includes pharmaceutically acceptable excipients; Preferably, the excipients include at least one of a carrier, a protective agent, an adjuvant, a formaldehyde, or a diluent; Preferably, the excipients include Tween, white oil, and Span.

10. The method for preparing the vaccine according to claim 9, characterized in that, Includes the following steps: The duck reovirus described in claim 6 is cultured and proliferated in cells to obtain a viral solution; or the duck reovirus is inoculated into specific pathogen-free duck embryos, incubated, and the allantoic fluid of the surviving embryos is collected to obtain a viral solution. Inactivation, resulting in an inactivated vaccine.

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