A method for inactivating viruses and uses thereof

Abstract

The present application relates to the technical field of biological medicine, and particularly discloses a virus inactivation method and application. The virus inactivation method of the present application uses formaldehyde as an inactivator, and further includes a formaldehyde blocking step after the virus inactivation with formaldehyde. The active ingredients of the blocking agent used in the formaldehyde blocking step are sodium bisulfite and EDTA-2Na, and the mass ratio of the sodium bisulfite and the EDTA-2Na is (3.8-4.2):1. The method of the present application can effectively reduce the toxicity of formaldehyde when formaldehyde is used as an inactivator, and reduce the residual amount of formaldehyde, and is particularly suitable for the inactivation of bovine dermatophilus virus and the blocking of formaldehyde.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a method for inactivating virus and application. BACKGROUND

[0002] Inactivation refers to the process of destroying the biological characteristics of pathogenic microorganisms while avoiding affecting their immunogenicity as much as possible. Common inactivators include formaldehyde, beta-propiolactone (BPL), BEI and other effective inactivators. However, not all viruses are suitable for all the above inactivators. Some viruses have better overall effect when inactivated by formaldehyde. However, there is a certain residual and toxicity in the formaldehyde solution used in the inactivation process, and there are few methods to solve this problem at present. Only a small amount of literature shows that sodium thiosulfate solution can neutralize formaldehyde, but in actual application, there are still problems of formaldehyde residue and toxicity that are difficult to solve. A small amount of literature shows that saturated sodium bisulfite solution can neutralize formaldehyde, but in order to achieve the ideal neutralization effect, the reaction temperature required by the neutralization condition is relatively high (some literature suggests about 60℃, and some literature suggests above 45℃) and the concentration of sodium bisulfite (saturated solution) required is relatively high. The above two conditions are not conducive to the preservation of the immunogenicity of the virus, and have limited applicability.

[0003] Lumpy skin disease (LSD) is a disease of cattle caused by Lumpy skin disease virus (LSDV) of the Poxviridae family. The clinical manifestations are fever, skin edema and local hard nodules or ulcers. It is widely spread, and it is necessary to quickly research a vaccine to prevent the disease and avoid new problems caused by the use of inactivators. SUMMARY

[0004] One of the purposes of the present application is to provide a method for inactivating virus with formaldehyde, which has little formaldehyde residue and toxicity. In addition, the present application also provides an inactivated vaccine with good immunogenicity and high safety based on the method.

[0005] The technical solution of the present application is as follows:

[0006] A method for inactivating virus, using formaldehyde as an inactivator, after inactivating virus with formaldehyde, further comprising a formaldehyde blocking step, the active ingredients of the blocking agent used in the formaldehyde blocking step are sodium bisulfite and EDTA-2Na, and the mass ratio of sodium bisulfite and EDTA-2Na is (3.8-4.2):1.

[0007] The application researches and finds that when formaldehyde is used as an inactivator for virus inactivation, the formaldehyde can be effectively removed by the specific blocking agent, thereby avoiding the influence of residual formaldehyde on the immunization effect and safety of subsequent preparations. Moreover, the blocking agent of the application can maintain effective formaldehyde blocking effect after being stored for 3 months, and has a long effective period.

[0008] In the formaldehyde blocking step of the method of the application, the pH value of the virus solution inactivated by formaldehyde is adjusted to 4.0-5.0, the temperature is adjusted to 35-37℃, and then the blocking agent is mixed to obtain a mixed solution; preferably, the mixing is performed by continuously stirring at 35-37℃ for 50-70 min.

[0009] The application finds that when the pH value condition of the blocking reaction is specifically limited, the influence on the immunization activity of the virus can be avoided, and the blocking agent can be effectively used (reacted with formaldehyde to remove formaldehyde) at a low temperature, thereby improving the processing efficiency, avoiding the influence of long-term reaction at the pH value on the inactivated virus, and further avoiding the adverse influence of high temperature on the inactivated virus.

[0010] Further preferably, in the formaldehyde blocking step, after the pH value of the virus solution inactivated by formaldehyde is adjusted to 4.0-5.0 and the temperature is adjusted to 35-37℃, the solution is first aerated and stirred, then the aeration is stopped, and then the blocking agent is mixed to obtain a mixed solution; preferably, the aeration rate is 0.8-1.2 L / min, and the stirring time is 20-40 min.

[0011] The application preferably adjusts the temperature and pH value before the virus solution inactivated by formaldehyde is contacted with the blocking agent, and then adds the step of aerating and stirring, so that the free gas formaldehyde is first carried out of the system with the gas flow and discharged from the reaction system, thereby better ensuring the subsequent processing effect.

[0012] In the method of the application, the mass ratio of the active ingredient of the blocking agent to formaldehyde used for virus inactivation is greater than 1:1, and is preferably (1-3.5):1, so that the formaldehyde is sufficiently removed, and the cost and solvent residue are avoided.

[0013] In the method of the application, before the pH value and temperature of the virus solution inactivated by formaldehyde are adjusted, the step of filtering the virus solution inactivated by formaldehyde after being stored at low temperature for 24 h or more is further included; the low temperature is 2-8℃, and is preferably 2-4℃; and the pore size of the filter material used in the filtering is 0.45-1 microns.

[0014] The present application preferably carries out low-temperature treatment on the formaldehyde-inactivated virus liquid for a certain time before the blocking agent treatment, which can make part of the formaldehyde in the form of trioxymethylene precipitate, and then removed from the solution system through subsequent filtration steps, which is beneficial to achieve better formaldehyde treatment effect. In addition, the filtration step can also remove the protein precipitate in the system, further improving the quality of the inactivated product.

[0015] In the method of the present application, the pH value of the obtained mixed solution is adjusted to 6.0-6.5, and the DO value is adjusted and maintained at 70%-90%, and the DO value is maintained for 1-2h; preferably, the DO value is maintained by aeration, and stirring operation is carried out at the same time, and the stirring speed is 60-120rpm;

[0016] Or, the pH value of the obtained mixed solution is adjusted to 5.0-5.5, the DO value is adjusted and maintained at 70%-90%, and the sodium thiosulfate is mixed, and the DO value is maintained for 1-2h; preferably, the DO value is maintained by aeration, and stirring operation is carried out at the same time, and the stirring speed is 60-120rpm.

[0017] It is found that after the reaction of the blocking agent and formaldehyde is completed, the pH value and the dissolved oxygen content of the mixed solution system are adjusted, and after a period of time, the residual amount of formaldehyde in the system is further reduced, and the pH value of the system is more moderate, which is beneficial to ensure the immunogenicity of the inactivated virus. Moreover, through aeration operation, the gas formaldehyde separated from the system can be carried away again, further ensuring the formaldehyde removal effect.

[0018] Preferably, while maintaining aeration, an aqueous solution of sodium thiosulfate is also added, which can further reduce the residual formaldehyde in combination with the corresponding system pH value.

[0019] In the method of the present application, the mass ratio of the sodium thiosulfate to the formaldehyde used for virus inactivation is (0.5-4):4, preferably (0.5-2):4, and more preferably (0.5-1.25):4, which can sufficiently remove the residual formaldehyde, and also consider the cost and solvent residue problems.

[0020] In the method of the present application, the virus is preferably bovine dermal nodular disease virus.

[0021] In the method of the present application, the conditions for virus inactivation with formaldehyde are as follows: the final concentration of formaldehyde in the inactivation mixed solution is 0.056%-0.08%, the inactivation mixed solution includes formaldehyde solution and virus liquid; the inactivation temperature is 30℃-37℃, preferably 35℃-37℃; the inactivation time is 35-40 hours, preferably 48 hours or more.

[0022] The concentration of formaldehyde in the inactivation mixed solution defined in the present application can make formaldehyde contact with viruses sufficiently and effectively, and ensure the inactivation effect.

[0023] In the process of researching the inactivated vaccine of bovine nodular dermatitis, through various tests, it is found that only formaldehyde and β-propiolactone can effectively inactivate the virus among the commonly used inactivators. However, the use of β-propiolactone has problems such as high production cost and strict inactivation conditions, so formaldehyde is preferably used for inactivating the bovine nodular dermatitis virus. However, there is a certain residual and toxicity in the formaldehyde solution used in the inactivation process, and there are few methods to solve this problem at present, and only a small amount of literature shows that sodium thiosulfate solution can neutralize formaldehyde, but the inventors found that it is difficult to solve the problem of formaldehyde residue and toxicity whether in accordance with the known method or by changing various conditions on the basis of the known method. In addition, high concentration of sodium bisulfite and high reaction temperature cannot be used for bovine nodular dermatitis virus (not conducive to the maintenance of virus immunogenicity).

[0024] Specifically, the inventors changed the reaction temperature and the concentration of sodium bisulfite and other conditions on the basis of the known method during the research and development, and found that under the premise of ensuring the immunogenicity of the virus (i.e. the reaction temperature is not higher than 37℃ and the concentration is not too high), the problem of formaldehyde toxicity cannot be solved, in other words, the known method using sodium bisulfite cannot be applied to the formaldehyde blocking in the field of virus inactivation. On the other hand, using the addition reaction principle of sodium sulfite and formaldehyde is not to completely remove formaldehyde, but to change its existing mode, and the inventors found through multiple tests that the residual amount of formaldehyde did not change significantly. Therefore, there are two difficulties in solving the problem of formaldehyde residue, i.e. the toxicity problem and the residual amount problem of formaldehyde. Based on the characteristics of bovine nodular dermatitis virus and the existing technical difficulties, the inventors finally found that when the inactivation method of the present application is applied, the inactivation effect can be ensured, the toxicity of formaldehyde can be effectively reduced, and the residual amount of formaldehyde can be reduced, which can further improve the product quality and safety of the corresponding vaccine, and also provides a theoretical basis, operation specification and guidance for the subsequent research and development of bovine nodular dermatitis inactivated vaccine.

[0025] As a specific embodiment of the present application, the method of the present application comprises:

[0026] S1 formaldehyde inactivation: take LSDV antigen solution, accurately add formaldehyde solution (40% formaldehyde) to the LSDV antigen solution according to a certain concentration, inactivate at 30℃-37℃ for 48h, and obtain virus solution inactivated completely.

[0027] S2 Formaldehyde Blocking: The completely inactivated virus solution from S1 was stored at 2–4°C for 24 hours. Then, after filtration through a depth filter at this temperature, it was heated to 35–37°C while adjusting the pH to 4.0–5.0. Sterile compressed air (1 L / min) was introduced and stirred thoroughly for 0.5 hours. After stirring, the aeration was stopped, and then an appropriate amount of blocking agent (a mixed solution of sodium bisulfite and EDTA-2Na) was added. After stirring continuously for 1 hour, the pH was adjusted to 6.0–6.5, and the dissolved oxygen (DO) value was adjusted and maintained at 70%–90%. The mixture was stirred at 60–120 rpm for 2 hours while maintaining the DO value. Finally, the pH was adjusted to 7.0–7.4 to obtain the final antigen solution.

[0028] Alternatively, the formaldehyde blocking method is as follows: S2.1: Take the completely inactivated virus solution from S1 and store it at 2-4℃ for 24 hours. Then, filter it using a depth filter at this temperature, heat it to 35-37℃, adjust the pH to 4.0-5.0, and introduce sterile compressed air (1L / min). Stir thoroughly for 0.5 hours, then stop the aeration. Next, add an appropriate amount of blocking agent (a mixed solution of sodium bisulfite and EDTA-2Na), stir continuously for 1 hour, adjust the pH to 5.0-5.5, adjust and maintain the DO value at 70%-90%, and stir at 60-120 rpm. Add an appropriate amount of sodium thiosulfate solution, stir continuously, maintain the DO value for 1-2 hours, and then adjust the pH to 7.0-7.4 to obtain the final antigen solution.

[0029] In step S1, the formaldehyde inactivation process involves adding a formaldehyde solution (40% formaldehyde) of 0.15% or more, and may further be 0.15% to 0.2%.

[0030] The final concentration of the blocking agent (a mixed solution of sodium bisulfite and EDTA-2Na) in step S2 is 2% to 10%, preferably 5% to 10%. When the blocking agent concentration is 8.8%, the amount of the blocking agent added should be greater than or equal to 120 μL / 10 ml, preferably 140 to 160 μL / 10 ml.

[0031] In step S2.1, the concentration of the sodium thiosulfate solution is 5% to 8%. When the concentration is 5%, the amount added is preferably greater than or equal to 40 μL / 10 mL.

[0032] The present invention also provides the application of the above method in reducing formaldehyde residue and / or toxicity in inactivated viruses, or in the preparation of inactivated vaccines.

[0033] The present invention also provides an inactivated vaccine, wherein the inactivated virus included in the inactivated vaccine is prepared by the above method.

[0034] The beneficial effects of this invention are at least as follows:

[0035] The virus inactivation process provided by this invention includes a formaldehyde blocking method, which can effectively inactivate viruses, is stable, simple to operate, and scalable, while effectively reducing the toxicity of formaldehyde and reducing the amount of formaldehyde residue. Inactivated vaccines produced by this method are safer, improve the quality of inactivated vaccine products, and are suitable for large-scale industrial production. It provides theoretical basis, operating specifications, and guidance for the development of inactivated vaccines (such as inactivated vaccines for bovine nodular dermatitis).

[0036] The method of this invention is particularly applicable to bovine nodular dermatitis virus, filling a gap in the industry regarding virus inactivation processes and blocking methods. Attached Figure Description

[0037] Figure 1 The inactivation curves of LSDV at 25°C for different formaldehyde concentrations in Example 1 of this invention are shown.

[0038] Figure 2 The inactivation curves of LSDV at 30℃ for different formaldehyde concentrations in Example 1 of the present invention are shown.

[0039] Figure 3 The inactivation curves of LSDV at 37°C for different formaldehyde concentrations in Example 1 of this invention are shown.

[0040] Figure 4 This is a cytotoxicity curve for different reaction times in Example 1 of the present invention.

[0041] Figure 5 This is a cytotoxicity curve of different concentrations of the blocking agent in Example 1 of the present invention.

[0042] Figure 6 Cytotoxicity curves at different reaction times in Comparative Example 1 of this invention.

[0043] Figure 7 Cytotoxicity curves for different reaction times in Comparative Example 2 of this invention.

[0044] Figure 8 Cytotoxicity curves for different reaction times in Comparative Example 3 of this invention.

[0045] Figure 9 Cytotoxicity curves for different reaction times in Comparative Example 4 of this invention.

[0046] Figure 10 Cytotoxicity curves for different reaction times in Comparative Example 5 of this invention. Detailed Implementation

[0047] The embodiments are implemented based on the technical solution of the present invention, providing detailed implementation methods and specific operation processes. These embodiments will help in understanding the present invention. The preferred embodiments of the present invention will be described in detail below with reference to the embodiments. It should be understood that the following embodiments are given for illustrative purposes only and are not intended to limit the scope of the present invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and purpose.

[0048] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0049] The inactivation test procedure involved in the specific implementation of this invention is as follows: Inactivation solution was inoculated into 4 good MDBK monolayer cells at a ratio of 25% (v / v) (2.5 ml added to each T25 cell flask). The cells were incubated at 37°C for 1 hour. The inactivation solution was then discarded, and 10 ml of cell maintenance medium was added per flask. Two controls were set up using the same method: one flask contained normal cells, and the other contained formaldehyde-containing maintenance medium (formaldehyde content consistent with the inactivation sample). The cells were incubated at 37°C in a 5% CO2 incubator for 6 days. CPE was observed daily, and the results were recorded. After 6 days of incubation, the culture was harvested. The harvested culture was then frozen and thawed, and MDBK cells were blindly passaged at 25% (v / v) for two passages. CPE was observed daily, and the results were recorded.

[0050] The procedure for plotting the inactivation curve is as follows: Take all inactivation test results, record the presence of CPE as positive (or "+"), and the absence of CPE as negative (or "-"). Plot the inactivation efficacy curve with inactivation time on the x-axis and the inactivation test positivity rate (lesion rate) on the y-axis.

[0051]

[0052] The specific procedure for cytotoxicity testing is as follows: the sample to be tested is serially diluted 10-fold with DMEM solution, and 10... -1 ~10 -5 Dilutions were prepared, and each dilution was seeded into a 96-well plate monolayer of MDBK cells (or other cell types, such as Vero cells or BHK cells). 21Four wells each of DMEM culture medium containing 2% newborn calf serum (NDBK) cells (one or more of sheep testicular cells, or any combination thereof with MDBK cells) were prepared, with 100 μl added to each well. The wells were incubated at 37°C for 1 hour for adsorption. Then, 100 μl of DMEM culture medium containing 2% newborn calf serum was added to each well. Four wells of normal cell control were prepared, with 100 μl of DMEM added to each well, followed by 100 μl of DMEM culture medium containing 2% newborn calf serum. The wells were incubated at 37°C in a 5% CO2 incubator for 6 days. Cell morphology changes were observed under a microscope, and the number of wells with abnormal cells at each dilution was recorded.

[0053] In this article, abnormal cell wells refer to abnormal cell phenomena such as cytoplasmic shrinkage, vacuoles, shedding, or even death caused by reagents contained in the tested sample.

[0054] The formaldehyde residue was tested according to the methods specified in the Chinese Veterinary Pharmacopoeia.

[0055] Example 1: Formaldehyde inactivation process and blocking method for bovine nodular dermatitis virus

[0056] This embodiment provides a method for inactivating viruses according to the present invention, specifically providing a formaldehyde inactivation process and blocking method for bovine nodular dermatitis virus (LSDV), mainly including: formaldehyde inactivation and formaldehyde blocking. Details are as follows:

[0057] 1.1 Formaldehyde inactivation LSDV process:

[0058] Take the LSDV antigen solution and divide it into several portions. Add formaldehyde solution (40% formaldehyde) to each LSDV antigen solution (10g) at different proportions. 7.5 TCID 50 Mix the formaldehyde solution in a solution of 0.05%, 0.1%, 0.15%, and 0.2% (v / v) at a concentration of 0.05%, 0.1%, 0.15%, and 0.2% (v / v), respectively. Transfer the thoroughly mixed samples into new centrifuge tubes for inactivation at 25℃, 30℃, and 37℃, shaking once every 2 hours or continuously. Take samples every 1–2 hours for inactivation testing and plot inactivation curves. Results are shown below. Figure 1 , Figure 2 , Figure 3 The formaldehyde concentration in the figure refers to the concentration of formaldehyde solution (40% formaldehyde) in the inactivation mixture.

[0059] The results show that the inactivation effect of formaldehyde on LSDV is related to the inactivation temperature. The inactivation temperature can be selected from 30℃ to 37℃, preferably 35℃ to 37℃.

[0060] The inactivation effect of formaldehyde on LSDV is related to the formaldehyde concentration. It is recommended to select a formaldehyde solution (40% formaldehyde) concentration of 0.15% or higher in the inactivation mixture, preferably 0.15% to 0.2%.

[0061] The inactivation effect of formaldehyde on LSDV is related to the inactivation time of formaldehyde. Generally, at a suitable concentration, LSDV can be completely inactivated within 35 to 40 hours. To ensure the inactivation effect, 48 hours or more is preferred.

[0062] The results also revealed some patterns at certain specific temperatures. At 25°C, formaldehyde solutions (40% formaldehyde) at various concentrations (0.05%–0.2%) in the inactivation mixture were insufficient to completely inactivate LSDV within 48 hours; further inactivation required an even longer time. See [link to relevant documentation]. Figure 1 At 30°C, a formaldehyde solution (40% formaldehyde) at a concentration of 0.05% in the inactivation mixture is unlikely to completely inactivate LSDV within 48 hours; a formaldehyde solution (40% formaldehyde) at a concentration of 0.1% in the inactivation mixture can completely inactivate LSDV within 46 hours; a formaldehyde solution (40% formaldehyde) at a concentration of 0.15% in the inactivation mixture can completely inactivate LSDV within 42 hours; and a formaldehyde solution (40% formaldehyde) at a concentration of 0.2% in the inactivation mixture can completely inactivate LSDV within 38 hours. See [link / reference] Figure 2 At 37°C, a formaldehyde solution (40% formaldehyde) at a concentration of 0.05% in the inactivation mixture is unlikely to completely inactivate LSDV within 48 hours; a formaldehyde solution (40% formaldehyde) at a concentration of 0.1% in the inactivation mixture can completely inactivate LSDV within 42 hours; a formaldehyde solution (40% formaldehyde) at a concentration of 0.15% in the inactivation mixture can completely inactivate LSDV within 38 hours; and a formaldehyde solution (40% formaldehyde) at a concentration of 0.2% in the inactivation mixture can completely inactivate LSDV within 34 hours. See Figure 3 .

[0063] Considering that large-scale virus inactivation often requires increased dosage of inactivating agent and doubled inactivation time to ensure inactivation effect, and based on the actual production of bovine nodular dermatitis inactivated vaccine, the virus inactivation temperature is determined to be 35℃~37℃, the concentration of inactivating agent (40% formaldehyde solution) is 0.15%~0.2%, and the inactivation time is 48 hours or more.

[0064] 1.2 Formaldehyde blocking:

[0065] First, the completely inactivated virus solution (formaldehyde solution (40% formaldehyde) concentration in the inactivation mixture was 0.2%) from "1.1" was stored at 4℃ for 24h. Then, it was filtered at this temperature using a depth filter (pore size of 0.45μm), heated to 36℃, and the pH was adjusted to 4.7. Sterile compressed air (1L / min) was introduced and stirred thoroughly for 0.5h. Then, the aeration was stopped. Subsequently, different volumes of blocking agent (see Table 1) were added to 10ml of the completely inactivated virus solution (inactivation mixture) treated as above. After stirring continuously for 1h, the pH was adjusted to 6.0, the DO value was adjusted to 85% by aeration, and the DO value was maintained by stirring continuously at 100rpm for 2h. Then, the pH was adjusted to 7.2, and cytotoxicity and formaldehyde residue were detected.

[0066] In this embodiment, the active ingredient in the blocking agent is a mixture of sodium bisulfite and EDTA-2Na in a mass ratio of 4:1, and the concentration of the blocking agent is 8.8%.

[0067] Cytotoxicity testing showed that the formaldehyde content in this embodiment was diluted by 10%. -2.5 ~10 ～3.0 Even after neutralization and blocking with 120 μL / 10 ml or higher of the above-mentioned inhibitors, the cytotoxicity was significantly reduced. See [link to relevant documentation]. Figure 4 The statistics show that, under different amounts of the aforementioned inhibitors, after a certain reaction time, abnormalities occurred at various sample dilutions.

[0068] The reduction in formaldehyde toxicity is related to the amount of inhibitor added. This example demonstrates that after neutralization and blocking with 120 μL / 10 ml or more of the aforementioned inhibitor, the toxicity of formaldehyde to cells is significantly reduced. See Figure 5 The statistics show that at different reaction times and with different amounts of inhibitors, abnormalities have occurred at different sample dilutions.

[0069] This embodiment also found that the reduction in formaldehyde content was related to the oxygen introduction time after the addition of the blocking agent; as time increased, the residual formaldehyde content decreased significantly. See Table 1. "No oxygen introduction" refers to adding the blocking agent, stirring for 1 hour, adjusting the pH to 7.2, and then performing the test.

[0070] Table 1 Summary of Formaldehyde Solution (40%) Residual Content Detection

[0071]

[0072] Example 2: Blocking method of formaldehyde inactivation process for bovine nodular dermatitis virus

[0073] In this embodiment, the completely inactivated virus solution (formaldehyde solution (40% formaldehyde) concentration in the inactivation mixture was 0.2%) from “1.1” of Example 1 was first stored at 4°C for 24 h. Then, after filtration at this temperature using a depth filter (pore size of 0.45 μm), it was heated to 36°C, and the pH was adjusted to 4.5. Sterile compressed air (1 L / min) was introduced and stirred thoroughly for 0.5 h. Then, the aeration was stopped. Next, 140 μL of the blocking agent from Example 1 was added to 10 ml of the completely inactivated virus solution treated above. After stirring continuously for 1 h, the pH was adjusted to 5.5, the DO value was adjusted to 70%, and the mixture was stirred at 100 rpm. Different volumes of sodium thiosulfate solution were added (see Table 2). After stirring continuously to maintain the DO value for 2 h, the pH was adjusted to 7.2, and cytotoxicity and formaldehyde residue were detected.

[0074] Unless otherwise specified, the conditions and reagents described in this embodiment are the same as those in Example 1.

[0075] In this embodiment, the concentration of sodium thiosulfate in the sodium thiosulfate solution is 5%.

[0076] This embodiment found that the reduction in formaldehyde content was related to the amount of sodium thiosulfate added and the oxygen introduction time after the addition of sodium thiosulfate. As the amount added increased, the residual formaldehyde content decreased significantly. See Table 2.

[0077] Table 2 Summary of Formaldehyde Solution (40%) Residual Content Detection

[0078]

[0079] Comparative Example 1

[0080] This comparative example provides a method for inactivating viruses, which is the same as that in Example 1, except that the active ingredient of the blocking agent is only sodium bisulfite.

[0081] The completely inactivated virus solution after formaldehyde blocking treatment was tested using the same method as in Example 1. The results showed that this method can also reduce the toxicity of formaldehyde, but the reduction effect is not as good as that of the method described in Example 1 (see Example 1). Figure 6 However, in terms of formaldehyde residue, it only had a slight effect and did not show any significant change (see Table 3).

[0082] Table 3 Summary of Formaldehyde Solution (40%) Residual Content Detection

[0083]

[0084] Comparative Example 2

[0085] This comparative example provides a method for inactivating viruses, which is the same as that in Example 1, except that the active ingredient of the blocking agent is sodium thiosulfate.

[0086] The virus solution, after being completely inactivated following the formaldehyde blocking step, was tested using the same method as in Example 1. The results showed that this method neither reduced the toxicity of formaldehyde nor reduced the residual amount of formaldehyde, as shown in Table 4. Figure 7 .

[0087] Table 4 Summary of Formaldehyde Solution (40%) Residual Content Detection

[0088]

[0089] Comparative Example 3

[0090] This comparative example provides a method for inactivating viruses, which is the same as that in Example 1, except that the active ingredients of the blocking agent are sodium bisulfite and EDTA-2Na, and the mass ratio of sodium bisulfite to EDTA-2Na is 3.5:1.

[0091] The completely inactivated virus solution after formaldehyde blocking treatment was tested using the same method as in Example 1. The results showed that this method can reduce the toxicity of formaldehyde (see Example 1). Figure 8 This method can also reduce the residual amount of formaldehyde (see Table 5), but it is slightly inferior to the method described in Example 1, namely, the ratio of the blocking agent described in Example 1 is better.

[0092] Table 5 Summary of Formaldehyde Solution (40%) Residual Content Detection

[0093]

[0094] Comparative Example 4

[0095] This comparative example provides a method for inactivating viruses, which is the same as that in Example 1, except that when formaldehyde is used for blocking, the completely inactivated virus solution is stored at low temperature, filtered, heated to 36°C, and the pH is adjusted to 6.0. The subsequent steps are the same as in Example 1.

[0096] The virus solution, after being completely inactivated following the formaldehyde blocking step, was tested using the same method as in Example 1. The results showed that the toxicity problem of formaldehyde could not be completely resolved (see Example 1). Figure 9 Moreover, the formaldehyde residue is relatively high (see Table 6).

[0097] Table 6 Summary of Formaldehyde Solution (40%) Residual Content Detection

[0098]

[0099] Comparative Example 5

[0100] This comparative example provides a method for inactivating viruses, which is the same as that in Example 1, except that in the formaldehyde blocking step, after adding the blocking agent and stirring continuously for 1 hour, the pH is adjusted to 4.5, the DO value is adjusted and maintained at 55% by ventilation, and the subsequent steps are the same as in Example 1.

[0101] The completely inactivated virus solution after formaldehyde blocking treatment was tested using the same method as in Example 1. The results showed that although this method can reduce the toxicity of formaldehyde (see Example 1), the virus is still inactivated. Figure 10 However, it cannot effectively reduce the residual amount of formaldehyde (see Table 7).

[0102] Table 7 Summary of Formaldehyde Solution (40%) Residual Content Detection

[0103]

[0104] Comparative Example 6

[0105] This comparative example provides a method for inactivating viruses, which is the same as that in Example 2, except that in the formaldehyde blocking step, after adding the blocking agent and stirring continuously for 1 hour, the pH is adjusted to 5.5, the DO value is adjusted and maintained at 60% by ventilation, and the subsequent steps are the same as in Example 2.

[0106] The virus solution that had been completely inactivated after the formaldehyde blocking step was tested using the same method as in Example 1. The results showed that the method could reduce both the toxicity and residual amount of formaldehyde (see Table 8). However, it was slightly inferior to the method described in Example 2, namely, the blocking agent and oxygenation treatment conditions of sodium thiosulfate described in Example 2 were better.

[0107] Table 8 Summary of Formaldehyde Solution (40%) Residual Content Detection

[0108]

[0109] Experimental example: immune challenge test

[0110] 1. Detection Method: Bovine nodular dermatitis virus treated with different methods was formulated into inactivated vaccines according to the same process (the mass ratio of inactivated virus solution to aluminum adjuvant was 1:1). Healthy susceptible cattle (age: 2-6 months, breed: Holstein) were then immunized twice according to the same immunization schedule (the interval between the first and second immunizations should be 21 days). 21 days after the second immunization, the cattle were challenged simultaneously with the OIE challenge protocol using bovine nodular dermatitis virus test virus that meets regulatory requirements. The incidence or protection status of all cattle was observed daily. If 4 / 5 or more of the immunized group (vaccinated group) showed protection, and 4 / 5 or more of the challenge control group (unvaccinated group) developed the disease, the test was considered qualified, indicating that the virus treated with the corresponding method or the prepared vaccine maintained good immunogenicity (good immunogenicity).

[0111] 2. Groups, 5 cows per group:

[0112] Formaldehyde inactivated group: Bovine nodular dermatitis virus was inactivated according to the inactivation method described in Example 1, 1.1, and then directly formulated into an inactivated vaccine without subsequent blocking operations. An immune challenge test was conducted to detect whether the method could maintain the immunogenicity of the virus.

[0113] Example 1 Group: Bovine nodular dermatitis virus was treated as described in 1.1 and 1.2 of Example 1 and formulated into an inactivated vaccine. An immune challenge test was conducted to detect whether the method could maintain the immunogenicity of the virus.

[0114] Example 2 group: Bovine nodular dermatitis virus was treated as described in Example 2 and formulated into an inactivated vaccine. An immune challenge test was conducted to detect whether the method could maintain the immunogenicity of the virus.

[0115] In each group, the specific inactivation conditions were as follows: the concentration of formaldehyde solution (40% formaldehyde) in the inactivation mixture was 0.15%, and inactivation was carried out at 36°C for 48 hours. The remaining unspecified details were consistent with those described in Example 1.

[0116] The specific blocking conditions in Example 1 were as follows: 120 μL of blocking agent was added to every 10 ml of inactivation mixture. Oxygen was introduced over a period of 2 hours after the addition of the blocking agent. All other unspecified details were consistent with those described in Example 1.

[0117] Challenge control group: Healthy susceptible cattle that have not been vaccinated were directly challenged to test the effectiveness of the virus strain.

[0118] 3. Criteria for determining the onset of bovine nodular dermatitis: Body temperature rises to 40℃ or higher and lasts for at least 3 days; after challenge, nodules appear on any part of the body surface except the injection site, and in severe cases, spread throughout the body; pathogen detection at the nodule sites, with PCR testing of nodules from secondary sites showing positive results for LSDV nucleic acid. If all three criteria are met, the disease can be diagnosed. Criteria for determining protection against bovine nodular dermatitis: Body temperature does not exceed 40.0℃; if the body temperature rises above 40.0℃, but remains above 40.0℃ for less than 3 days; after challenge, there are no obvious abnormalities in mental state or appetite, and no secondary nodules appear. If the above conditions are met, the animal can be considered protected.

[0119] The results of the immune challenge test are shown in Table 9.

[0120] Table 9. Protection against viral challenge

[0121]

[0122]

[0123] In summary, the inactivation process provided by this invention can effectively inactivate bovine nodular dermatitis virus, and the inactivated virus still maintains good immunogenicity (see Table 9). The blocking method provided by this invention does not require high-temperature reaction, and the method can effectively maintain the immunogenicity of bovine nodular dermatitis virus (see Table 9), and can also effectively reduce the toxicity of formaldehyde, with significant effects. The additional treatment process added after blocking can effectively reduce the residual amount of formaldehyde, and the results are stable after multiple measurements (the fluctuation range is stable within ±0.01%). The blocking method of bovine nodular dermatitis virus formaldehyde inactivation process provided by this invention is stable, scalable, simple to operate, and safer overall.

[0124] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for inactivating a virus, using formaldehyde as an inactivating agent, further comprising a formaldehyde blocking step after virus inactivation with formaldehyde, characterized in that, The active ingredients of the blocking agent used in the formaldehyde blocking step are sodium bisulfite and EDTA-2Na, and the mass ratio of sodium bisulfite to EDTA-2Na is (3.8-4.2):1; the virus is bovine nodular dermatitis virus.

2. The method according to claim 1, characterized in that, In the formaldehyde blocking step, the pH value of the virus solution after formaldehyde inactivation is first adjusted to 4.0-5.0, and the temperature is adjusted to 35-37°C. Then, it is mixed with the blocking agent to obtain a mixed solution.

3. The method according to claim 2, characterized in that, The mixing is carried out by continuous stirring at 35–37°C for 50–70 minutes.

4. The method according to claim 3, characterized in that, In the formaldehyde blocking step, the pH value of the virus solution after formaldehyde inactivation is adjusted to 4.0-5.0, and the temperature is adjusted to 35-37°C. Then, the solution is first aerated and stirred thoroughly. After the aeration is stopped, the solution is then mixed with the blocking agent to obtain a mixed solution.

5. The method according to claim 4, characterized in that, The ventilation rate is 0.8-1.2 L / min, and the stirring time is 20-40 min.

6. The method according to claim 1, characterized in that, The mass ratio of the active ingredient of the blocking agent to the formaldehyde used in virus inactivation is ≥1:

1.

7. The method according to claim 6, characterized in that, The mass ratio of the active ingredient of the blocking agent to the formaldehyde used in virus inactivation is (1-3.5):

1.

8. The method according to any one of claims 2-5, characterized in that, The mass ratio of the active ingredient of the blocking agent to the formaldehyde used in virus inactivation is ≥1:

1.

9. The method according to claim 8, characterized in that, The mass ratio of the active ingredient of the blocking agent to the formaldehyde used in virus inactivation is (1-3.5):

1.

10. The method according to any one of claims 2-5, 9, characterized in that, Before adjusting the pH and temperature of the formaldehyde-inactivated virus solution, the method further includes a step of storing the formaldehyde-inactivated virus solution at a low temperature for 24 hours or longer before filtering, wherein the low temperature is 2-8°C; and the pore size of the filter material is 0.45-1 micrometer during the filtration.

11. The method according to claim 8, characterized in that, Before adjusting the pH and temperature of the formaldehyde-inactivated virus solution, the method further includes a step of storing the formaldehyde-inactivated virus solution at a low temperature for 24 hours or longer before filtering, wherein the low temperature is 2-8°C; and the pore size of the filter material is 0.45-1 micrometer during the filtration.

12. The method according to claim 10, characterized in that, The low temperature is 2 to 4°C.

13. The method according to claim 11, characterized in that, The low temperature is 2 to 4°C.

14. The method according to any one of claims 2-5, 9, and 11-13, characterized in that, The method also includes adjusting the pH of the obtained mixed solution to 6.0-6.5 and adjusting and maintaining the DO value at 70%-90% for 1-2 hours. Alternatively, the mixture may include the steps of adjusting the pH of the obtained mixed solution to 5.0–5.5, adjusting and maintaining the DO value at 70%–90%, and mixing it with sodium thiosulfate, wherein the DO value is maintained for 1–2 hours.

15. The method according to claim 8, characterized in that, The method also includes adjusting the pH of the obtained mixed solution to 6.0-6.5 and adjusting and maintaining the DO value at 70%-90% for 1-2 hours. Alternatively, the mixture may include the steps of adjusting the pH of the obtained mixed solution to 5.0–5.5, adjusting and maintaining the DO value at 70%–90%, and mixing it with sodium thiosulfate, wherein the DO value is maintained for 1–2 hours.

16. The method according to claim 10, characterized in that, The method also includes adjusting the pH of the obtained mixed solution to 6.0-6.5 and adjusting and maintaining the DO value at 70%-90% for 1-2 hours. Alternatively, the mixture may include the steps of adjusting the pH of the obtained mixed solution to 5.0–5.5, adjusting and maintaining the DO value at 70%–90%, and mixing it with sodium thiosulfate, wherein the DO value is maintained for 1–2 hours.

17. The method according to claim 14, characterized in that, The DO value is maintained by ventilation, and stirring is performed while maintaining the DO value, with the stirring speed being 60-120 rpm.

18. The method according to claim 15 or 16, characterized in that, The DO value is maintained by ventilation, and stirring is performed while maintaining the DO value, with the stirring speed being 60-120 rpm.

19. The method according to claim 14, characterized in that, The mass ratio of sodium thiosulfate to formaldehyde used for virus inactivation is (0.5-4):

4.

20. The method according to any one of claims 15-17, characterized in that, The mass ratio of sodium thiosulfate to formaldehyde used for virus inactivation is (0.5-4):

4.

21. The method according to claim 18, characterized in that, The mass ratio of sodium thiosulfate to formaldehyde used for virus inactivation is (0.5-4):

4.

22. The method according to claim 19 or 21, characterized in that, The mass ratio of sodium thiosulfate to formaldehyde used for virus inactivation is (0.5-2):

4.

23. The method according to claim 20, characterized in that, The mass ratio of sodium thiosulfate to formaldehyde used for virus inactivation is (0.5-2):

4.

24. The method according to claim 19, 21 or 23, characterized in that, The conditions for inactivating the virus with formaldehyde are as follows: the final concentration of formaldehyde in the inactivation mixture is 0.056%-0.08%, and the inactivation mixture includes formaldehyde solution and virus solution; the inactivation temperature is 30℃~37℃; and the inactivation time is 35~40 hours.

25. The method according to claim 20, characterized in that, The conditions for inactivating the virus with formaldehyde are as follows: the final concentration of formaldehyde in the inactivation mixture is 0.056%-0.08%, and the inactivation mixture includes formaldehyde solution and virus solution; the inactivation temperature is 30℃~37℃; and the inactivation time is 35~40 hours.

26. The method according to claim 22, characterized in that, The conditions for inactivating the virus with formaldehyde are as follows: the final concentration of formaldehyde in the inactivation mixture is 0.056%-0.08%, and the inactivation mixture includes formaldehyde solution and virus solution; the inactivation temperature is 30℃~37℃; and the inactivation time is 35~40 hours.

27. The method according to claim 24, characterized in that, The inactivation temperature is 35℃~37℃; the inactivation time is 48 hours or more.

28. The method according to claim 25 or 26, characterized in that, The inactivation temperature is 35℃~37℃; the inactivation time is 48 hours or more.

29. The use of the method according to any one of claims 1-28 in reducing formaldehyde residue and / or toxicity in inactivated viruses, or in the preparation of inactivated vaccines.

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