A method for culturing human metapneumovirus

Abstract

The present application relates to the field of biomedical technology and discloses a method for culturing human metapneumovirus, comprising the following steps: preparing a culture medium and placing it in a culture bottle: the culture medium comprising: DMEM culture medium, glucose, sodium bicarbonate, HEPES buffer, chondroitin sulfate, hyaluronic acid; selecting host cells; preparing a culture fluid comprising: L-glutamine, bovine serum albumin, antibiotics, and N-acetylcysteine; and inoculating human metapneumovirus into the culture medium for adsorption and infection. The present invention adds chondroitin sulfate and hyaluronic acid to the culture medium, utilizing chondroitin sulfate and hyaluronic acid to bind to glycosaminoglycans on the surface of host cells to form a complex, thereby increasing the interaction sites between the virus and the host cells, thereby enhancing the expression of viral receptors on the surface of the host cells, optimizing the cell membrane environment, thereby improving the attachment and entry efficiency of hMPV and significantly promoting the rapid proliferation of the virus.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, in particular to a method for culturing human metapneumovirus. Background Art

[0002] Human-Metapneumovirus (hMPV) is a major respiratory pathogen with a widespread global distribution, causing acute respiratory infections, particularly in children, and of significant public health significance. Laboratory isolation and identification of hMPV primarily rely on cell culture techniques, while efficient viral amplification is a key prerequisite for related research and vaccine development.

[0003] Existing hMPV culture optimization strategies mostly focus on cell line selection, optimization of viral infection multiplicity, and adjustment of culture conditions. Although these methods have improved virus yield to a certain extent, their applicability is relatively narrow and it is difficult to significantly increase virus yield, which directly restricts the efficiency of virus isolation and identification and the progress of subsequent scientific research. Summary of the Invention

[0004] In response to the shortcomings of the existing technology, the present invention provides a method for culturing human metapneumovirus, which solves the problem that although the existing technology improves the virus yield to a certain extent, its applicability is narrow and it is difficult to significantly increase the virus yield, which restricts the efficiency of virus isolation and identification and the progress of subsequent scientific research work.

[0005] To achieve the above objectives, the present invention is implemented through the following technical solutions: A method for culturing human metapneumovirus, comprising the following steps:

[0006] Prepare a culture medium and fill it into a culture bottle: the culture medium includes the following components in parts by weight: DMEM medium: 800-950 parts, glucose: 1000-1200 parts, sodium bicarbonate: 800-1000 parts, HEPES buffer: 2000-2300 parts, chondroitin sulfate: 30-50 parts, hyaluronic acid: 10-40 parts;

[0007] Among them, DMEM medium provides cells with basic nutrients (such as amino acids and vitamins) to support the normal growth of host cells;

[0008] Glucose provides cells with energy substrates and can significantly enhance cellular metabolic activity during viral replication;

[0009] Sodium bicarbonate and HEPES buffer together maintain the pH stability of the culture environment. The buffering capacity of HEPES is particularly suitable for high-density cell culture;

[0010] Chondroitin sulfate and hyaluronic acid can serve as components of the extracellular matrix, forming a glycosaminoglycan-like network structure on the cell surface, simulating the microenvironment of natural viral receptors, and promoting human metapneumovirus (hMPV) to attach to and enter host cells more efficiently, thereby significantly improving the infection efficiency.

[0011] Select host cells and inoculate them into culture medium for primary culture;

[0012] Among them, HEp-2 cells are an epithelial cell line with a clear origin and good sensitivity to hMPV, making them suitable as a host for viral infection; controlling the appropriate cell density can maintain a balance between cell spacing and nutrient supply, prevent excessive fusion or insufficient nutrition, and provide sufficient cell surface receptor sites for virus adsorption.

[0013] Prepare a culture solution and add it to a culture bottle. The culture solution includes the following components in parts by weight: 150-200 parts of L-glutamine, 30-70 parts of bovine serum albumin, 100-120 parts of antibiotics, and 170-200 parts of N-acetylcysteine;

[0014] Among them, L-glutamine is an important nitrogen source and energy supplier, which can enhance cell metabolic activity and support efficient viral replication;

[0015] Bovine serum albumin (BSA) can stabilize the culture environment, reduce toxin adsorption, and promote the stable existence of virus particles in the culture system;

[0016] Antibiotics (penicillin and streptomycin) inhibit bacterial contamination and ensure pure virus growth;

[0017] N-acetylcysteine (NAC) has antioxidant function, can remove ROS (reactive oxygen species), improve the stability of cell membrane structure, and cooperate with the complex formed by chondroitin sulfate and hyaluronic acid to further optimize cell membrane permeability and promote the fusion and entry of viruses into cell membranes.

[0018] Human metapneumovirus was inoculated into the culture medium for adsorption and infection.

[0019] Preferably, the culture medium is prepared and placed in a culture bottle, and after the culture medium components are placed in the same container, they are stirred at a temperature of 25 to 37° C. and a rotation speed of 150 to 200 rpm for 10 to 15 minutes, and then placed in the culture bottle.

[0020] Preferably, the host cells include HEp-2 cells, and the host cell density is: 1×10 5 ~1×10 6 cells / mL, the volume ratio of the host cells to the culture medium is: 1-10:10-100, and the hyaluronic acid includes sodium hyaluronate.

[0021] Among them, HEp-2 cells are an epithelial cell line with a clear origin and good sensitivity to hMPV, making them suitable as a host for viral infection; controlling the appropriate cell density can maintain a balance between cell spacing and nutrient supply, prevent excessive fusion or insufficient nutrition, and provide sufficient cell surface receptor sites for virus adsorption.

[0022] Preferably, the initial culture is carried out at a temperature of 36-38° C. and 5% CO 2 for 2-3 hours.

[0023] Among them, this condition simulates the physiological environment in the body, which is conducive to the cells entering a stable growth period and adjusting their state, helping to achieve the optimal physiological state before virus inoculation and enhance the ability to respond to the virus.

[0024] Preferably, in the preparation of the culture medium, L-glutamine, bovine serum albumin, antibiotics and N-acetylcysteine are mixed and stirred at a temperature of 36 to 38° C. and a rotation speed of 200 to 280 rpm for 10 to 16 minutes.

[0025] This process can promote the full and uniform mixing of the components, while enhancing the solubility and stability of NAC at a suitable temperature, ensuring that it continues to play an antioxidant and membrane stabilizing role during subsequent viral infection.

[0026] Preferably, the antibiotics include penicillin and streptomycin, and the mass ratio of the two is 1-1.2:1-1.2.

[0027] Among them, this ratio can effectively prevent bacterial contamination and ensure the stability of the culture system without inhibiting the activity of host cells.

[0028] Preferably, the concentration of human metapneumovirus in the inoculation is 1×10 3 ~1×10 5 PFU / mL, and the volume ratio of the human metapneumovirus to the host cells is 1-10:10-100.

[0029] Among them, controlling the virus inoculation concentration helps achieve synchronous infection and prevent experimental errors caused by too high or too low MOI (multiplicity of infection); a reasonable volume ratio ensures that the virus particles can fully contact the cell surface receptors and increase the infection initiation rate.

[0030] Preferably, the adsorption and infection are carried out at a temperature of 36-38° C. and 5% CO 2 for 1.5-2.5 hours.

[0031] This condition can simulate the physiological environment in the body, which is conducive to the cells entering a stable growth period and adjusting their state, helping to achieve the optimal physiological state before virus inoculation and enhance the ability to respond to the virus.

[0032] The present invention provides a method for culturing human metapneumovirus, which has the following beneficial effects:

[0033] 1. The present invention adds chondroitin sulfate and hyaluronic acid to the culture medium, utilizing the fact that chondroitin sulfate and hyaluronic acid can bind to glycosaminoglycans on the surface of host cells to form a complex, thereby increasing the interaction sites between the virus and the host cells, thereby enhancing the expression of viral receptors on the surface of the host cells, optimizing the cell membrane environment, thereby improving the attachment and entry efficiency of hMPV and significantly promoting the rapid proliferation of the virus.

[0034] 2. The present invention adds N-acetylcysteine to the culture medium and uses NAC as an antioxidant. It can provide sulfhydryl groups to reduce free radicals, thereby achieving the purpose of scavenging free radicals in cells, maintaining the redox balance in cells, protecting cells from oxidative damage, and enhancing the fluidity of cell membranes, thereby reducing the damage of oxidative stress to cells, thereby improving cell vitality and stability, and promoting the interaction between viruses and host cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Schematic diagram of the method of the present invention. DETAILED DESCRIPTION

[0036] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the drawings in the specification of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0037] In order to better understand the present invention, the above contents are described in detail below in conjunction with specific embodiments.

[0038] Please see the attached Figure 1 :

[0039] Example 1

[0040] Prepare 900 parts of DMEM medium, 1150 parts of glucose, 950 parts of sodium bicarbonate, 2100 parts of HEPES buffer, 40 parts of chondroitin sulfate, and 25 parts of hyaluronic acid (sodium hyaluronate). Stir at 32°C and 180 rpm for 13 minutes, then transfer to a culture flask.

[0041] The host cells (HEp-2, cell density of 5×10 5cells / mL) and inoculated into culture medium for primary culture (volume ratio of host cells to culture medium was 5:70). Primary culture conditions were as follows: transfer the culture flask to an incubator and culture at 37°C and 5% CO2 for 2.3 h.

[0042] Prepare culture medium: 175 parts L-glutamine, 55 parts bovine serum albumin, 115 parts antibiotics (penicillin:streptomycin = 1:1.1), and 180 parts N-acetylcysteine. Stir at 37°C and 240 rpm for 14 minutes and add to the culture flask.

[0043] Human metapneumovirus (concentration: 1×10 4 The culture flask was then transferred to an incubator for 2 h of adsorption and infection at 37 °C and 5% CO2.

[0044] Example 2

[0045] Prepare 800 parts of DMEM medium, 1000 parts of glucose, 800 parts of sodium bicarbonate, 2000 parts of HEPES buffer, 30 parts of chondroitin sulfate, and 10 parts of hyaluronic acid (sodium hyaluronate). Stir at 25°C and 150 rpm for 10 minutes, then transfer the cells into a culture flask.

[0046] The host cells (HEp-2, cell density of 1×10 5 cells / mL) and inoculated into culture medium for primary culture (volume ratio of host cells to culture medium was 1:100). Primary culture conditions were as follows: transfer the culture flask to an incubator and culture at 37°C and 5% CO2 for 2 h.

[0047] Prepare culture medium: 150 parts L-glutamine, 30 parts bovine serum albumin, 100 parts antibiotics (penicillin:streptomycin = 1:1.2), and 170 parts N-acetylcysteine. Stir at 37°C and 200 rpm for 10 minutes and add to the culture flask.

[0048] Human metapneumovirus (concentration: 1×10 3 The culture flask was then transferred to an incubator for adsorption and infection at 37°C and 5% CO2 for 1.5 h.

[0049] Example 3

[0050] Prepare 950 parts of DMEM medium, 1200 parts of glucose, 1000 parts of sodium bicarbonate, 2300 parts of HEPES buffer, 50 parts of chondroitin sulfate, and 40 parts of hyaluronic acid (sodium hyaluronate). Stir at 37°C and 200 rpm for 15 minutes, then transfer the cells to a culture flask.

[0051] The host cells (HEp-2, cell density of 1×10 6 cells / mL) and inoculated into culture medium for primary culture (volume ratio of host cells to culture medium was 10:90). Primary culture conditions: transfer the culture flask to an incubator and culture at 37°C and 5% CO2 for 3 h.

[0052] Prepare culture medium: 200 parts L-glutamine, 70 parts bovine serum albumin, 120 parts antibiotics (penicillin:streptomycin = 1.2:1), and 200 parts N-acetylcysteine. Stir at 37°C and 280 rpm for 16 minutes and add to the culture flask.

[0053] Human metapneumovirus (concentration: 1×10 5 The culture flask was then transferred to an incubator for adsorption and infection at 37°C and 5% CO2 for 2.5 h.

[0054] Comparative Example 1:

[0055] Based on Example 1, chondroitin sulfate was omitted and the rest remained the same.

[0056] Comparative Example 2:

[0057] Based on Example 2, 20 parts of chondroitin sulfate were used, and the rest remained the same.

[0058] Comparative Example 3:

[0059] Based on Example 3, 60 parts of chondroitin sulfate were used, and the rest remained the same.

[0060] Comparative Example 4:

[0061] Based on Example 1, hyaluronic acid (sodium hyaluronate) is omitted, and the rest remain the same.

[0062] Comparative Example 5:

[0063] Based on Example 2, hyaluronic acid (sodium hyaluronate) is 9 parts, and the rest remains the same.

[0064] Comparative Example 6:

[0065] Based on Example 3, 50 parts of hyaluronic acid (sodium hyaluronate) were used, and the rest remained the same.

[0066] Comparative Example 7:

[0067] Based on Example 1, without hyaluronic acid (sodium hyaluronate) and chondroitin sulfate, the rest remain the same.

[0068] Comparative Example 8:

[0069] Based on Example 1, without N-acetylcysteine, the rest remain the same.

[0070] Comparative Example 9:

[0071] Based on Example 2, 160 parts of N-acetylcysteine were used, and the rest remained the same.

[0072] Comparative Example 10:

[0073] Based on Example 3, 210 parts of N-acetylcysteine were used, and the rest remained the same.

[0074] Experiment 1:

[0075] Purpose of the experiment

[0076] This study aimed to investigate the effects of chondroitin sulfate and hyaluronic acid on enhancing host cell (HEp-2) sensitivity to human metapneumovirus (hMPV) and promoting rapid viral proliferation. By comparing viral titers, cell viability, and cell number under different conditions (including varying the amount of chondroitin sulfate and hyaluronic acid added), the key roles of these two components in hMPV culture were clarified.

[0077] Sample setup: Examples 1 to 3, Comparative Examples 1 to 7.

[0078] Data Collection:

[0079] 1. Virus Titer (PFU / mL) Determination

[0080] Equipment: cell culture incubator (37°C, 5% CO2), microscope (for observing cytopathic effect, CPE), multichannel pipette, 6-well or 12-well plates (for viral titer determination), sterile PBS buffer, cell stain (neutral red).

[0081] Method: Plaque-Assay

[0082] Steps:

[0083] Cell inoculation: HEp-2 cells were plated at 1×10 5 The cells were seeded at a density of 10 cells / mL in 6-well plates and cultured at 37°C and 5% CO2 for 24 h to allow the cells to adhere to the wall and form a monolayer.

[0084] Virus dilution: The virus sample was diluted 10-fold in sterile PBS buffer (10 -1 , 10 -2 , 10 -3 ).

[0085] Virus inoculation: Aspirate the culture medium from the 6-well plate, add 100 µL of diluted virus solution to each well, shake gently to evenly distribute the virus, and allow to adsorb for 1 h at 37°C and 5% CO2.

[0086] Cover with agar: Gently add agar medium (containing 2% low-melting point agar and 1% fetal bovine serum) preheated to 42°C to each well to cover the cell layer. After the agar solidifies, transfer the 6-well plate to a 37°C, 5% CO2 incubator and culture for 5 days.

[0087] Staining and counting: remove the agar layer, stain the cell monolayer with neutral red, and observe and count the number of plaques.

[0088] Calculate virus titer: Calculate virus titer (PFU / mL) based on the number of plaques and dilution factor.

[0089] 2. Cell Viability (%) Determination

[0090] Equipment: cell culture incubator (37°C, 5% CO2), microplate reader (for absorbance measurement), 96-well plate, cell counting kit.

[0091] Method: CCK-8 method.

[0092] Steps:

[0093] Cell inoculation: HEp-2 cells were plated at 1×10 4 The cells were seeded at a density of 10 cells / well in a 96-well plate and cultured at 37°C, 5% CO2 for 24 h to allow the cells to adhere to the wall.

[0094] Reagent addition: Add 10µL CCK-8 reagent to each well and continue incubation for 4 hours.

[0095] Stop the reaction and measure the absorbance directly.

[0096] Absorbance determination: The absorbance was measured at a wavelength of 450 nm using a microplate reader.

[0097] Calculate cell viability: Calculate cell viability (%) based on the absorbance value. The formula is:

[0098] Cell viability (%) = (absorbance of experimental group / absorbance of control group) × 100%.

[0099] 3. Cell Number Determination

[0100] Equipment: cell counting chamber, microscope.

[0101] Method: Cell counting method.

[0102] Steps:

[0103] Cell cultures were harvested on days 3 and 7 after virus adsorption infection.

[0104] After diluting the cell suspension, take 10µL and add it to a cell counting plate, and count the number of cells under a microscope.

[0105] Calculate the number of cells per unit volume (mL).

[0106] The test results are shown in Table 1:

[0107] Table 1:

[0108]

[0109] Experimental Summary

[0110] From the experimental data, it can be seen that the virus titers of Examples 1 to 3 are significantly higher than those of Comparative Examples 1 to 7, indicating that the addition of chondroitin sulfate and hyaluronic acid significantly enhances the sensitivity of host cells to hMPV and promotes the rapid proliferation of the virus. Specific analysis is as follows:

[0111] Virus titers of Examples 1 to 3:

[0112] The virus titer of Example 1 was 1.2×10 6 PFU / mL, Example 2 is 8.7×10 5 PFU / mL, Example 3 is 1.5×10 6 PFU / mL, which were significantly higher than the virus titers of Comparative Examples 1 to 7.

[0113] This indicates that adding chondroitin sulfate and hyaluronic acid to the culture medium can significantly improve the proliferation efficiency of hMPV.

[0114] Virus titers of Comparative Example 1 and Comparative Example 4:

[0115] The virus titer of Comparative Example 1 (without chondroitin sulfate) was 6.3×10 4 PFU / mL decreased by about 80% compared with Example 1.

[0116] The virus titer of Comparative Example 4 (without hyaluronic acid) was 5.9×10 4 PFU / mL decreased by about 85% compared with Example 1.

[0117] This shows that after completely removing chondroitin sulfate or hyaluronic acid, the virus titer was significantly reduced, indicating that these two components play a key role in the virus adsorption and infection process.

[0118] Virus titers of Comparative Example 2 and Comparative Example 5:

[0119] The virus titer of Comparative Example 2 (20 parts of chondroitin sulfate) was 7.8×10 4 PFU / mL decreased by about 93% compared with Example 1.

[0120] The virus titer of Comparative Example 5 (9 parts of hyaluronic acid) was 6.7×10 4 PFU / mL decreased by about 94% compared with Example 1.

[0121] This shows that after reducing the content of chondroitin sulfate or hyaluronic acid, the viral titer is also significantly reduced, further verifying the importance of these two components.

[0122] Virus titers of Comparative Example 3 and Comparative Example 6:

[0123] The virus titer of Comparative Example 3 (60 parts of chondroitin sulfate) was 1.1×10 5 PFU / mL decreased by about 90% compared with Example 1.

[0124] The virus titer of Comparative Example 6 (50 parts of hyaluronic acid) was 9.2×10 4 PFU / mL decreased by about 93% compared with Example 1.

[0125] This shows that after increasing the content of chondroitin sulfate or hyaluronic acid, the virus titer increased, but the increase was limited, indicating that there is an optimal range for the addition of these two ingredients.

[0126] Virus titer of Comparative Example 7:

[0127] The virus titer of Comparative Example 7 (without chondroitin sulfate and hyaluronic acid) was 4.8×10 4 PFU / mL decreased by about 96% compared with Example 1.

[0128] At the same time, the cell viability of Comparative Example 7 also decreased significantly (81.5%), indicating that the synergistic effect of these two components is crucial for viral proliferation and cell protection.

[0129] Moreover, the cell numbers of Examples 1 to 3 reached 3.8×10 5 / mL and 7.2×10 5 / mL, which is 2 to 3 times higher than that of comparative examples 1 to 7, further verifying the promoting effect of chondroitin sulfate and hyaluronic acid on viral proliferation.

[0130] This suggests that chondroitin sulfate and hyaluronic acid bind to glycosaminoglycans on the host cell surface, forming a complex that increases the number of interaction sites between the virus and the host cell, thereby optimizing the cell membrane environment and enhancing the efficiency of hMPV attachment and entry. This effect significantly increases host cell sensitivity to hMPV and promotes rapid viral proliferation. Furthermore, the synergistic effect of chondroitin sulfate and hyaluronic acid further enhances the efficiency and stability of viral culture, providing important technical support for the research and application of hMPV.

[0131] Experiment 2:

[0132] Purpose of the experiment

[0133] This study aimed to investigate the sensitivity of N-acetylcysteine to human metapneumovirus (hMPV) and its ability to promote rapid viral proliferation. By comparing viral titers and cell viability under different conditions (varying the amount of N-acetylcysteine added), we determined the key roles of these two components in hMPV culture.

[0134] Sample settings: Examples 1 to 3, Comparative Examples 8 to 10.

[0135] Data Collection:

[0136] 1. Virus Titer (PFU / mL) Determination

[0137] Equipment: cell culture incubator (37°C, 5% CO2), microscope (for observing cytopathic effect, CPE), multichannel pipette, 6-well or 12-well plates (for viral titer determination), sterile PBS buffer, cell stain (neutral red).

[0138] Method: Plaque-Assay

[0139] Steps:

[0140] Cell inoculation: HEp-2 cells were plated at 1×10 5 The cells were seeded at a density of 10 cells / mL in 6-well plates and cultured at 37°C and 5% CO2 for 24 h to allow the cells to adhere to the wall and form a monolayer.

[0141] Virus dilution: The virus sample was diluted 10-fold in sterile PBS buffer (10 -1 , 10 -2 , 10 -3 ).

[0142] Virus inoculation: Aspirate the culture medium from the 6-well plate, add 100 µL of diluted virus solution to each well, shake gently to evenly distribute the virus, and allow to adsorb for 1 h at 37°C and 5% CO2.

[0143] Cover with agar: Gently add agar medium (containing 2% low-melting point agar and 1% fetal bovine serum) preheated to 42°C to each well to cover the cell layer. After the agar solidifies, transfer the 6-well plate to a 37°C, 5% CO2 incubator and culture for 5 days.

[0144] Staining and counting: remove the agar layer, stain the cell monolayer with neutral red, and observe and count the number of plaques.

[0145] Calculate virus titer: Calculate virus titer (PFU / mL) based on the number of plaques and dilution factor.

[0146] 2. Cell Viability (%) Determination

[0147] Equipment: cell culture incubator (37°C, 5% CO2), microplate reader (for absorbance measurement), 96-well plate, cell counting kit.

[0148] Method: CCK-8 method.

[0149] Steps:

[0150] Cell inoculation: HEp-2 cells were plated at 1×10 4 The cells were seeded at a density of 10 cells / well in a 96-well plate and cultured at 37°C, 5% CO2 for 24 h to allow the cells to adhere to the wall.

[0151] Reagent addition: Add 10µL CCK-8 reagent to each well and continue incubation for 4 hours.

[0152] Stop the reaction and measure the absorbance directly.

[0153] Absorbance determination: The absorbance was measured at a wavelength of 450 nm using a microplate reader.

[0154] Calculate cell viability: Calculate cell viability (%) based on the absorbance value. The formula is:

[0155] Cell viability (%) = (absorbance of experimental group / absorbance of control group) × 100%.

[0156] 3. Cell Number Determination

[0157] Equipment: cell counting chamber, microscope.

[0158] Method: Cell counting method.

[0159] Steps:

[0160] Cell cultures were harvested on days 3 and 7 after virus adsorption infection.

[0161] After diluting the cell suspension, take 10µL and add it to a cell counting plate, and count the number of cells under a microscope.

[0162] Calculate the number of cells per unit volume (mL).

[0163] The test results are shown in Table 2:

[0164] Table 2:

[0165]

[0166] Experimental Summary

[0167] From the experimental data, it can be seen that the virus titer, cell viability, and cell number of Examples 1 to 3 are significantly higher than those of Comparative Examples 8 to 10, indicating that the addition of N-acetylcysteine significantly enhances the sensitivity of host cells to hMPV and promotes the rapid proliferation of the virus. The specific analysis is as follows:

[0168] The virus titers of Examples 1 to 3 were 1.2×10 6 PFU / mL, 8.7×10 5 PFU / mL and 1.5×10 6 PFU / mL, which is significantly higher than the virus titer of Comparative Examples 8 to 10. At the same time, the cell number of Examples 1 to 3 reached 3.4×10 5 / mL and 3.9×10 5 / mL, reaching 6.8×10 5 / mL and 7.4×10 5 / mL, which is 2 to 3 times higher than that of comparative examples 8 to 10, further verifying the promoting effect of N-acetylcysteine on viral proliferation.

[0169] After completely removing N-acetylcysteine in Comparative Example 8, viral titer decreased by approximately 40%, and cell counts also decreased significantly, indicating that N-acetylcysteine plays a key role in viral adsorption and infection. After reducing the content of N-acetylcysteine in Comparative Example 9, viral titer and cell counts also decreased significantly, further verifying the importance of this ingredient. After increasing the content of N-acetylcysteine in Comparative Example 10, viral titer and cell counts increased, but the increase was limited, indicating that there is an optimal range for the addition of N-acetylcysteine.

[0170] N-acetylcysteine scavenges free radicals, maintains intracellular redox balance, protects cells from oxidative damage, and enhances cell membrane fluidity, further promoting interaction between the virus and host cells. This effect significantly improves the proliferation efficiency and culture stability of hMPV, providing important technical support for the research and application of hMPV.

[0171] Therefore, in summary, chondroitin sulfate, hyaluronic acid, and N-acetylcysteine exert a synergistic effect in hMPV culture, significantly improving viral proliferation efficiency and culture stability. Chondroitin sulfate and hyaluronic acid bind to glycosaminoglycans on the surface of host cells to form a complex, increasing the interaction sites between the virus and host cells, thereby optimizing the cell membrane environment and improving the attachment and entry efficiency of hMPV. N-acetylcysteine protects cells from oxidative damage by scavenging free radicals and maintaining intracellular redox balance, while also enhancing cell membrane fluidity, further promoting the interaction between the virus and host cells. The synergistic effect of these three components significantly enhances the host cell's sensitivity to hMPV and promotes rapid viral proliferation.

[0172] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A method for culturing human metapneumovirus, characterized in that: The following steps are involved: Prepare a culture medium and fill it into a culture bottle: the culture medium includes the following components in parts by weight: DMEM medium: 800-950 parts, glucose: 1000-1200 parts, sodium bicarbonate: 800-1000 parts, HEPES buffer: 2000-2300 parts, chondroitin sulfate: 30-50 parts, hyaluronic acid: 10-40 parts; Select host cells and inoculate them into culture medium for primary culture; Prepare a culture solution and add it to a culture bottle. The culture solution includes the following components in parts by weight: 150-200 parts of L-glutamine, 30-70 parts of bovine serum albumin, 100-120 parts of antibiotics, and 170-200 parts of N-acetylcysteine; Human metapneumovirus was inoculated into the culture medium for adsorption and infection; The host cells are HEp-2 cells, and the host cell density is: 1×10 5 ~1×10 6 cells / mL, the volume ratio of the host cells to the culture medium is: 1-10:10-100, and the hyaluronic acid is sodium hyaluronate; The concentration of human metapneumovirus in the inoculation was 1×10 3 ~1×10 5 PFU / mL, and the volume ratio of the human metapneumovirus to the host cells is 1-10:10-100.

2. The method for culturing human metapneumovirus according to claim 1, wherein: The culture medium is prepared and placed in a culture bottle. After the culture medium components are placed in the same container, they are stirred for 10 to 15 minutes at a temperature of 25 to 37° C. and a rotation speed of 150 to 200 rpm, and then placed in the culture bottle.

3. The method for culturing human metapneumovirus according to claim 1, characterized in that: The initial culture is carried out at a temperature of 36-38° C. and 5% CO 2 for 2-3 hours.

4. The method for culturing human metapneumovirus according to claim 1, characterized in that: In the culture medium preparation, L-glutamine, bovine serum albumin, antibiotics and N-acetylcysteine are mixed and stirred at a temperature of 36 to 38° C. and a rotation speed of 200 to 280 rpm for 10 to 16 minutes.

5. The method for culturing human metapneumovirus according to claim 1, characterized in that: The antibiotics include penicillin and streptomycin, and the mass ratio of the two is 1-1.2:1-1.

2.

6. The method for culturing human metapneumovirus according to claim 1, characterized in that: The adsorption and infection are carried out at a temperature of 36 to 38° C. and 5% CO 2 for 1.5 to 2.5 hours.

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