Influenza virus drug resistance analysis method and application

Abstract

The application discloses an influenza virus drug resistance analysis method and application. The influenza virus drug resistance analysis method comprises the following steps: incubating an anti-influenza virus drug with an influenza virus to obtain a drug-virus incubation mixture; inoculating the drug-virus incubation mixture into host cells; culturing the inoculated host cells; allowing the virus to infect the cells and replicate; mixing the cultured cells with a cell proliferation activity detection reagent; evaluating the inhibition degree of the drug on the virus replication according to the cell proliferation activity; and judging the drug resistance of the influenza virus. The application evaluates the inhibition degree of the drug on the virus replication by the proliferation of the infected cells, simulates the dynamic process of the virus infection in the body, and can reflect the actual drug efficacy. The application is suitable for all anti-influenza virus drugs, is simple and objective and reliable to operate, and can be widely applied to the drug resistance detection of the influenza virus.

Description

[0001] This application claims priority to patent application number 2026109391709 (the earlier application was filed on June 26, 2026, and is entitled "A Method for Analyzing Drug Resistance of Influenza Virus and Its Application"). Technical Field

[0002] This invention belongs to the field of biotechnology and relates to a method and application for analyzing influenza virus drug resistance. Background Technology

[0003] Influenza viruses are the main pathogens causing seasonal influenza, with influenza B virus being one of the important circulating strains. Neuraminidase inhibitors (NAIs) are the main drugs used clinically to treat influenza B virus and are a class of antiviral drugs recommended by the World Health Organization (WHO) for the prevention and treatment of influenza B virus infection. Neuraminidase (NA) is a glycoprotein distributed on the envelope of the influenza virus. It catalyzes the hydrolysis of sialic acid, assisting mature influenza viruses in detaching from host cells and releasing viral particles to infect new cells. NAIs effectively inhibit viral replication and spread by inhibiting neuraminidase activity, blocking the uncoating and release of the virus from the surface of infected cells, and simultaneously preventing the spread of viral particles from respiratory mucus to other cells.

[0004] The existing methods for detecting influenza virus drug resistance and their limitations are mainly as follows: 1. Methods such as qPCR and gene chips mainly detect known resistance sites and cannot detect unknown or new resistance sites. While whole-genome sequencing can discover new mutations, it relies on existing annotation databases to analyze their functions, and unknown or new mutations may not actually affect drug target activity. 2. The NAI assay is based on quantifying the inhibitory effect of drugs on viral NA activity, directly assessing the sensitivity of influenza virus to NAI drugs. This assay is only applicable to NAI and cannot assess viral resistance to other types of antiviral drugs, potentially missing resistance caused by non-NA gene mutations. Furthermore, the NAI assay only tests in vitro enzyme activity and cannot reflect the dynamic infection process within cells. 3. The plaque subtraction method determines the minimum inhibitory concentration of a drug by observing the inhibition of plaque formation in infected cells by the drug in the upper layer of agar. The plaque count is a result of visual judgment, which involves subjective factors. Different operators may have different interpretations of the results, which is not conducive to the standardization of the method. The cytopathic effect inhibition assay assesses viral resistance by observing the degree to which a drug inhibits the cytopathic effect of a virus-infected cell. However, this is also a visual assessment and therefore subjective.

[0005] Therefore, there is an urgent need to provide a new method for analyzing influenza virus drug resistance to solve the problems of existing technologies, such as failure to reflect actual drug efficacy, narrow applicability, and strong subjectivity. Summary of the Invention

[0006] To address the shortcomings of existing technologies and practical needs, this invention provides a method and application for analyzing influenza virus drug resistance. It dynamically links cytotoxicity detection with viral replication, enabling functional verification of drug resistance and overcoming the deficiency that simple gene sequencing cannot reflect actual drug efficacy. This becomes a key supporting technology for the clinical application of anti-influenza drugs.

[0007] To achieve this objective, the present invention employs the following technical solution: In a first aspect, the present invention provides a method for analyzing influenza virus drug resistance, the method comprising: co-incubating an anti-influenza virus drug with an influenza virus to obtain a drug-virus incubation mixture; inoculating the drug-virus incubation mixture into host cells; culturing the inoculated host cells to allow the virus to infect the cells and replicate; mixing the cultured cells with a cell proliferation activity detection reagent; assessing the degree of inhibition of viral replication by the drug based on cell proliferation activity; and determining the drug resistance of the influenza virus.

[0008] This invention assesses the degree of drug inhibition of viral replication by observing the proliferation of infected cells, simulating the dynamic process of viral infection in vivo and reflecting the actual efficacy of the drug; this invention is applicable to all antiviral drugs.

[0009] Preferably, the influenza virus includes any one or a combination of at least two of the following: influenza A virus, influenza B virus, influenza C virus, or influenza D virus.

[0010] Preferably, the host cells include MDCK cells and / or MDCK-STX cells.

[0011] Preferably, the co-incubation temperature is 35-38℃ (e.g., 35℃, 36℃, 37℃ or 38℃), and the time is 1-3 h (e.g., 1 h, 2 h or 3 h).

[0012] Preferably, the culture temperature is 30-40℃ (e.g., 30℃, 35℃, 37℃ or 40℃), and the culture time is 36-60 h (e.g., 36 h, 48 h or 60 h).

[0013] Preferably, the cell proliferation activity detection reagent includes CCK-8 reagent.

[0014] The CCK-8 (Cell Counting Kit-8) reagent can be used for simple and accurate cell proliferation and toxicity analysis. The CCK-8 reagent contains the water-soluble tetrazolium salt WST-8, which, in the presence of an electron coupling agent, can be reduced by mitochondrial dehydrogenases to a highly water-soluble orange-yellow formazan product. For the same cells, the color intensity is linearly related to the number of viable cells; that is, the OD value detected by the microplate reader is linearly related to the number of viable cells. Therefore, this characteristic can be used for cell proliferation and toxicity analysis.

[0015] Preferably, the evaluation of the drug's inhibitory effect on viral replication includes: calculating the viral inhibition rate, and calculating the drug's half-maximal inhibitory concentration (IC50) based on the viral inhibition rate. 50 ; The formula for calculating the virus inhibition rate is shown in equation (1); Inhibition rate = (OD value of experimental wells - mean OD value of virus control wells) / (mean OD value of cell control wells - mean OD value of virus control wells) Equation (1).

[0016] In this invention, the reading wavelength requirements for the microplate are: main wavelength 450 nm and reference wavelength 620 nm.

[0017] Preferably, determining the drug resistance of the influenza virus includes: setting the half-maximal inhibitory concentration (IC50) of the virus to be tested at an IC50 value. 50 Value and reference virus half-maximal inhibitory concentration (IC50) 50 The values ​​are compared, and the judgment criteria are as follows: When the half-inhibitory concentration (IC50) of the virus to be detected 50 Value < 10 times the reference plant's half-maximal inhibitory concentration (IC50) 50 When the value is [value missing], it is determined to be sensitive to the drug; When the half-maximal inhibitory concentration (IC50) of the reference plant was 10 times that of the reference plant... 50 Value ≤ IC50 of the virus to be detected 50 Value ≤ 100 times the reference plant's half-maximal inhibitory concentration (IC50) 50 When the value is [value missing], it is determined to be a decrease in drug sensitivity; When the half-inhibitory concentration (IC50) of the virus to be detected 50 Value > 100 times the reference plant's half-maximal inhibitory concentration (IC50) 50 When the value is [value missing], it is determined to be a highly reduced sensitivity to the drug.

[0018] Preferably, when the half-maximal inhibitory concentration (IC50) of the virus to be detected is... 50 Value ≥ 10 times the reference plant's half-maximal inhibitory concentration (IC50) 50 When the value is reached, at least three independent repeated tests should be performed, and the average value should be used as the final judgment basis.

[0019] Preferably, the influenza virus drug resistance analysis method further includes: performing a half-maximum tissue culture infectious dose (TCID50) test on the virus sample before co-incubating the anti-influenza drug with the influenza virus to determine the viral infection titer.

[0020] Preferably, the half-maximal tissue culture infection dose (TCID50) is calculated using the Reed-Muench method.

[0021] Preferably, the anti-influenza virus drug comprises any one or a combination of at least two of the following: neuraminidase inhibitors, PA protein inhibitors, or PB2 subunit inhibitors of RNA polymerase.

[0022] Secondly, the present invention provides the application of the influenza virus drug resistance analysis method described in the first aspect in the preparation of products for clinical efficacy evaluation of anti-influenza virus drugs.

[0023] Preferably, the influenza virus includes any one or a combination of at least two of the following: influenza A virus, influenza B virus, influenza C virus, or influenza D virus.

[0024] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention assesses the degree of drug inhibition on viral replication by measuring the proliferation of infected cells, simulating the dynamic process of viral infection in vivo, and can reflect the actual efficacy of the drug at the cellular level, making up for the deficiency that simple gene sequencing cannot reflect the actual efficacy. (2) This invention assesses viral drug resistance by detecting the proliferation of infected cells, rather than by detecting neuraminidase activity. It is applicable to all anti-influenza virus drugs, including neuraminidase inhibitors and other novel antiviral drugs, and solves the problem that existing neuraminidase inhibitor drug trials are only applicable to neuraminidase inhibitor drugs. Detailed Implementation

[0025] To further illustrate the technical means and effects of this invention, the following embodiments are provided for further explanation. It should be understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0026] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0027] Example 1 This embodiment provides a method for analyzing drug resistance in influenza B virus.

[0028] The samples to be tested were two clinically derived nasopharyngeal swab samples.

[0029] 1. Equipment, reagents and consumables 1.1 The equipment is shown in Table 1.

[0030] Table 1 1.2 The reagents are shown in Table 2.

[0031] Table 2 1.3 Reagent Preparation 1.3.1 MDCK-STX cell growth medium: Add 5 mL Pen-Strep and 50 mL FBS to 445 mL DMEM / F12 solution, mix well, and prepare MDCK-STX cell growth medium containing 1% Pen-Strep and 10% FBS. Store at 2-8℃ for one month.

[0032] 1.3.2 Virus growth medium: Add 0.5 mL Pen-Strep, 1.25 mL HEPES buffer, 1.25 mL 7.5% bovine serum albumin component V and 0.05 mL TPCK-trypsin (2 mg / mL) to 46.95 mL DMEM / F12 solution to prepare a virus growth medium with a final TPCK-trypsin concentration of approximately 2 μg / mL. Store at 2-8℃ for one month.

[0033] 1.3.3 Virus diluent: Prepare a virus diluent with a final TPCK-trypsin concentration of approximately 2 μg / mL according to the ratio of DMEM / F12:7.5% bovine serum albumin component: Pen Strep: 2 mg / mL TPCK-trypsin = 857:132:10:1. Store at 2-8℃ for one month.

[0034] 2. Quality Control 2.1 Viral suspension TCID 50 Testing and Quality Control Using a known TCID 50 Fluorine-concentrated influenza virus solution was used as a positive control on each plate, and virus dilution was used as a negative control. The deviation of the positive control test results from their labeled results was within ±1 lg TCID. 50 / mL indicates a positive control in control; no cytopathic effect was observed in all wells of the negative control, indicating a negative control in control. Simultaneous control of both positive and negative controls in control indicates an acceptable result for this study.

[0035] 2.2 Quality Control of Drug Resistance Analysis and Testing Each plate includes a cell control and a corresponding virus control for each sample. A mean OD value of the cell control minus a mean OD value of the corresponding virus control for each sample ≥ 1.000 indicates that both the cell control and the virus control for that sample are in control.

[0036] 2.3 The reference virus strain and the sample to be tested must be of the same subtype.

[0037] 2.4 If the experimental results of the selected dilution factor are calculated in GraphPad Prism 8, the IC will be... 50 If the results are significantly inconsistent with the experimental results, the dilution factor should be adjusted and the experiment repeated until the IC50 value is reached. 50 The results are accurate.

[0038] 3. Operating Procedures 3.1 Sample preparation 3.1.1 Sample filtration: To avoid interference from bacteria and other microorganisms in sample detection, the sample must be filtered through a 0.45 μm sterile syringe filter.

[0039] 3.1.2 Sample activation: Take 1 mL of filtered sample solution and add 1 μL of 2 mg / mL TPCK-trypsin.

[0040] 3.2 Viral replication 3.2.1 Take out the corresponding number of T25 cell culture flasks according to the sample quantity and number them; 3.2.2 Cell suspension preparation: MDCK-STX cells in the logarithmic growth phase were collected and a sufficient amount of cell suspension was prepared using cell growth medium (cell density approximately 3.0 × 10⁻⁶). 5 ~5.0×10 5 (cells / mL) 3.2.3 Cell culture: Add 5 mL of cell suspension to each T25 cell culture flask and culture at 37°C and 5% CO2 for about 24 h (cell growth density to about 70%~90%). 3.2.4 Sample addition: Discard the cell growth medium in the cell culture flask and wash the cells three times with 2 mL PBS. Add 1 mL of virus sample, gently shake, and incubate at 37°C in a 5% CO2 incubator for 2 h for adsorption. 3.2.5 Medium change: Aspirate the sample, wash the cells twice with 2 mL PBS, add 8 mL of virus growth medium, shake gently, and incubate at 35°C in a 5% CO2 incubator; 3.2.6 Virus capture: Observe the cytopathic effect daily. Harvest the virus suspension when 75% to 100% of the cells show cytopathic effects. Even if there are no cytopathic effects, the virus should be harvested on the 7th day of culture.

[0041] 3.2.7 Note: 3.2.7.1 If the culture medium in the sample does not contain TPCK-trypsin, which is conducive to the adsorption of virus particles onto host cells, it should be filtered first, and then TPCK-trypsin should be added.

[0042] 3.2.7.2 Virus suspension harvesting: The proliferated virus suspension was freeze-thawed twice, then centrifuged at 4℃ and 5000 rpm for 30 min. The supernatant was collected, filtered through a 0.45 μm sterile syringe filter, and 8 μL of 2 mg / mL TPCK-trypsin was added. After mixing, the mixture was aliquoted and stored at ≤-80℃ for later use.

[0043] 3.3 TCID 50 Detection 3.3.1 Take out the corresponding number of 96-well cell culture plates according to the number of samples, and number them; 3.3.2 Cell suspension preparation: MDCK-STX cells in the logarithmic growth phase were collected and a sufficient amount of cell suspension was prepared using cell growth medium (cell density approximately 1×10⁻⁶). 5 ~2×10 5 (cells / mL) 3.3.3 Cell plating: Add 0.1 mL of cell suspension to each well of a 96-well plate and incubate at 37°C and 5% CO2 for about 24 h (to achieve a cell growth density of about 80%~90% per well). 3.3.4 Virus suspension sample dilution: The sample to be tested is serially diluted 10-fold to 10 using virus diluent. -6 A total of 7 samples with different titer levels were obtained (10 times). 0 ~10 -6 Meanwhile, the positive control was diluted at 7 different titer levels (10). -1 ~10 -7 The negative control was a virus dilution. In each 96-well plate, two virus suspension samples and one positive control were tested. An independent negative control was set up for each sample, with four wells for each dilution and four wells for the negative control. 3.3.5 Sample addition: Discard the culture medium in the 96-well plate and wash once with PBS. Add 0.1 mL of diluted sample solution or virus dilution and incubate at 35℃ and 5% CO2 for 48 h. 3.3.6 Microscopic recording results: The number of pores in the lesion cells was observed using a research-grade inverted fluorescence microscope; 3.3.7 Calculate the TCID of the sample using the Reed-Muench method. 50 The formula is as follows: ; 3.3.7.1 lgTCID 50 / 0.1 mL = Distance ratio × Difference between the logarithms of dilutions + Logarithm of dilutions with a lesion rate higher than 50% 3.3.7.2 lgTCID 50 / mL = Distance ratio × Difference between the logarithms of dilutions + Logarithm of dilutions with a lesion rate higher than 50% - 1 3.3.8 Note: The sample dilution method is: 50 μL of the test sample + 450 μL of virus diluent.

[0044] 3.4 Drug resistance analysis and detection 3.4.1 Take out the corresponding number of 96-well cell culture plates according to the number of samples, and number them; 3.4.2 Cell suspension preparation: MDCK-STX cells in the logarithmic growth phase were collected and a sufficient amount of cell suspension was prepared using cell growth medium (cell density approximately 1×10⁻⁶). 5 ~2×10 5 (cells / mL) 3.4.3 Cell plating: Add 0.1 mL of cell suspension to each well of a 96-well plate and incubate at 37°C and 5% CO2 for about 24 h (to achieve a cell growth density of about 80% to 90% per well). 3.4.4 Drug Preparation: Weigh an appropriate amount of the drug and prepare a stock solution with a concentration of 3.52 nmol / μL according to the drug mass (mg): 80% ethanol volume (mL) ratio of 21:13. Then prepare according to Table 3 below: Table 3 3.4.5 Drug dilution: Based on experimental requirements, assess and select appropriate dilution ratios (such as 3x, 4x, 5x, 6x, and 7x) and dilute the drug solution to eight concentrations using virus diluent. 3.4.6 Virus dilution: The TCID of each virus suspension sample was measured according to step 3.3. 50 Take a sufficient amount of the harvested virus suspension from each sample, mix well, and dilute each virus suspension to the appropriate multiple (100 TCID50) using virus diluent. 50 / 0.05 mL-400 TCID 50 / 0.05 mL); 3.4.7 96-well plate setup: Take a new 96-well plate (plate A) and set up cell control, test sample, reference virus strain, and virus control (each test sample and reference virus strain has its corresponding virus control). The cell control has 12 wells, the test sample and the reference virus strain each have 3 wells, and the test sample and the reference virus strain each have 4 wells. 3.4.8 Virus drug incubation: Add 80 μL of the virus solution corresponding to the test sample and 80 μL of virus diluent to the virus control wells, respectively. Add 80 μL of the virus solution corresponding to the test sample and 80 μL of the drug solution of the corresponding concentration to the test wells, respectively. In addition, add 160 μL of virus diluent to the cell control. Mix well and incubate at 37°C for 2 h. 3.4.9 Virus inoculation: After incubation, wash the 96-well cell culture plate (B plate) with a cell density of 80%-90% with PBS. Transfer 100 μL of solution from each well of plate A to the corresponding well of plate B and incubate at 35°C and 5% CO2 for 48 h. 3.4.10 Preparation of CCK-8 working solution: CCK-8 solution and MDCK-STX cell growth medium were mixed at a ratio of 1:10 to prepare CCK-8 working solution; 3.4.11 After the B-plate virus inoculation culture is completed, add 150 μL PBS to each well to wash the cell plate twice, aspirate the PBS, add 110 μL CCK-8 working solution to each well, and continue to incubate at 37℃ and 5% CO2 for 2 h; 3.4.12 Plate Reading: After incubation, the absorbance of each well was measured using a microplate reader with a main wavelength of 450 nm and a reference wavelength of 620 nm; the virus inhibition rate of each sample was calculated, and the IC50 of each sample was calculated using GraphPad Prism 8. 50 The absorbance values ​​measured by the ELISA reader are shown in Table 4.

[0045] Table 4 Note 3.4.13: 3.4.13.1 If the virus suspension contains lgTCID 50 If the concentration of the virus suspension is ≤ 2.0 mL, no dilution is required and it can be used directly; if the TCID of the virus suspension cannot be calculated... 50 If the sample is negative, then it cannot be tested for viral drug resistance.

[0046] 3.4.13.2 Virus inhibition rate = (OD value of experimental wells - mean OD value of virus control wells) / (mean OD value of cell control wells - mean OD value of virus control wells). The results of the virus inhibition rate are shown in Table 5.

[0047] Table 5 4. Result Judgment 4.1 When the virus to be detected is IC 50 Value < 10 times the reference strain IC 50 When the value is [value missing], it is judged as drug sensitivity; 4.2 When the reference strain IC is 10 times50 Value ≤ Detected virus IC 50 Value ≤ 100 times the reference strain IC 50 When the value is [value missing], it is judged as decreased drug sensitivity; 4.3 When the virus to be detected is IC 50 Value > 100 times that of the reference strain IC 50 When the value is [value missing], it is judged as a highly reduced sensitivity to drugs.

[0048] Note: If the virus to be detected is IC 50 Value ≥ 10 times the reference strain IC 50 When the value is reached, the virus requires three independent tests. If the average of the three test results is still ≥10 times the reference strain IC50 value, the test is considered successful. 50 If the value is not specified, it will be interpreted according to the corresponding standard (sensitivity is reduced).

[0049] 5. Experimental Results: Test sample N1 IC 50 Value (0.1440) < 10 times the IC50 of the reference virus strain 50 The value (0.1255) indicates sensitivity to the drug.

[0050] N2 IC of the test sample 50 Value (0.1502) < 10 times the IC50 of the reference virus strain 50 The value (0.1255) indicates sensitivity to the drug.

[0051] The experimental results are shown in Table 6.

[0052] Table 6 Example 2 This embodiment provides a method for analyzing influenza virus drug resistance.

[0053] The only difference between this embodiment and Embodiment 1 is the type of cells used. Embodiment 1 used MDCK-STX cells, while Embodiment 2 used MDCK cells.

[0054] 1. Equipment, reagents and consumables 1.1 The equipment is shown in Table 7: Table 7 1.2 The reagents are shown in Table 8: Table 8 1.3 Reagent Preparation 1.3.1 MDCK cell growth medium: Add 5 mL Pen-Strep and 50 mL FBS to 445 mL DMEM / F12 solution, mix well, and prepare MDCK cell growth medium containing 1% Pen-Strep and 10% FBS. Store at 2-8℃ for one month.

[0055] 1.3.2 Virus growth medium: Add 0.5 mL Pen-Strep, 1.25 mL HEPES buffer, 1.25 mL 7.5% bovine serum albumin component V and 0.05 mL TPCK-trypsin (2 mg / mL) to 46.95 mL DMEM / F12 solution to prepare a virus growth medium with a final TPCK-trypsin concentration of approximately 2 μg / mL. Store at 2-8℃ for one month.

[0056] 1.3.3 Virus dilution: Prepare a virus dilution with a final TPCK-trypsin concentration of approximately 2 μg / mL according to the ratio of DMEM / F12 : 7.5% bovine serum albumin component V : Pen Strep : 2 mg / mL TPCK-trypsin = 857 : 132 : 10 : 1. Store at 2-8℃ for one month.

[0057] 2. Quality Control 2.1 Virus suspension TCID 50 During the detection, a known TCID was used. 50 Fluorine virus solution was used as a positive control for each plate, and virus dilution was used as a negative control. The deviation of the positive control test results from their labeled results was within ±1lg TCID. 50 / mL indicates a positive control in control; no cytopathic effect was observed in all wells of the negative control, indicating a negative control in control. Simultaneous control of both positive and negative controls in control indicates an acceptable result for this study.

[0058] 2.2 In the drug resistance analysis, each plate included a cell control and a corresponding virus control for each sample. A mean OD value of the cell control minus a mean OD value of the corresponding virus control for each sample ≥ 1.000 indicates that both the cell control and the virus control for that sample are in control.

[0059] 2.3 The reference virus strain and the sample to be tested must be of the same subtype.

[0060] 2.4 If the experimental results of the selected dilution factor are used to calculate the IC in GraphPad Prism 8 50 If the results are significantly inconsistent with the experimental results, the dilution factor should be adjusted and the experiment repeated until the IC50 value is reached. 50 The results are accurate.

[0061] 3. Operation process 3.1 Sample preparation 3.1.1 Sample Filtration: To avoid interference from bacteria and other microorganisms in the sample detection, the sample must be filtered through a 0.45 μm sterile syringe filter before detection. 3.1.2 Sample activation: Take 1 mL of filtered sample solution and add 1 μL of 2 mg / mL TPCK-trypsin.

[0062] 3.2 Virus proliferation 3.2.1 Take out the corresponding number of T25 cell culture flasks according to the sample quantity, and number them; 3.2.2 Cell suspension preparation: Collect MDCK cells in the logarithmic growth phase and prepare a sufficient amount of cell suspension using cell growth medium (cell density approximately 3.0~5.0 × 10⁻⁶). 5 (cells / mL) 3.2.3 Cell culture: Add 5 mL of cell suspension to each T25 cell culture flask and culture at 37°C and 5% CO2 for about 24 h (cell growth density to about 70%~90%). 3.2.4 Sample addition: Discard the cell growth medium in the cell culture flask and wash the cells three times with 2 mL PBS. Add 1 mL of virus sample, gently shake, and incubate at 37°C in a 5% CO2 incubator for 2 h for adsorption. 3.2.5 Change medium: Aspirate the sample, wash the cells twice with 2 mL PBS, add 8 mL virus growth medium, shake gently, and incubate at 35℃ in a 5% CO2 incubator.

[0063] 3.2.6 Virus capture: Observe the cytopathic effect daily. Harvest the virus suspension when 75% to 100% of the cells show cytopathic effects. Even if there are no cytopathic effects, the virus should be harvested on the 7th day of culture.

[0064] 3.2.7 Virus suspension harvesting: The proliferated virus suspension was freeze-thawed twice, then centrifuged at 4℃ and 4700~5000 rpm for 30 min. The supernatant was collected, filtered through a 0.45 μm sterile syringe filter, and 8 μL of 2 mg / mL TPCK-trypsin was added. After mixing, the mixture was aliquoted and stored at ≤-80℃ for later use.

[0065] 3.3 TCID 50 Detection 3.3.1 Take out the corresponding number of 96-well cell culture plates (hereinafter referred to as 96-well plates) according to the number of samples, and number them; 3.3.2 Cell suspension preparation: Collect MDCK cells in the logarithmic growth phase and prepare a sufficient amount of cell suspension using cell growth medium (cell density approximately 1~2×10⁻⁶). 5 (cells / mL) 3.3.3 Cell plating: Add 0.1 mL of cell suspension to each well of a 96-well plate and incubate at 37°C and 5% CO2 for about 24 h (to achieve a cell growth density of about 80% to 90% per well). 3.3.4 Virus suspension sample dilution: The test samples were serially diluted 10-fold to 10⁶ times using virus diluent, resulting in 7 samples at different titer levels (10⁶). 0 ~10 -6 Meanwhile, the positive control was diluted at 7 different titer levels (10). -1 ~10 -7 The negative control was a virus dilution.

[0066] 3.3.5 In each 96-well plate, two virus suspension samples and one positive control were tested. An independent negative control was set up for each sample. Four wells were set up for each dilution, and four wells were also set up for the negative control. 3.3.6 Sample addition: Discard the culture medium in the 96-well plate and wash once with PBS. Add 0.1 mL of diluted sample solution or virus dilution and incubate at 35°C and 5% CO2 for 48 h. 3.3.7 Microscopic recording results: The number of pores of diseased cells was observed using a research-grade inverted fluorescence microscope; 3.3.8 Calculate the TCID of the sample using the Reed-Muench method. 50 The formula is as follows: lgTCID 50 / 0.1mL = Distance ratio × Difference between logarithms of dilutions + Logarithm of dilutions with a lesion rate higher than 50% lgTCID 50 / mL = Distance ratio × Difference between logarithms of dilutions + Logarithm of dilutions with a lesion rate higher than 50% - 1 3.4 Drug resistance analysis and detection 3.4.1 Take out the corresponding number of 96-well cell culture plates (hereinafter referred to as 96-well plates) according to the number of samples, and number them; 3.4.2 Cell suspension preparation: Collect MDCK cells in the logarithmic growth phase and prepare a sufficient amount of cell suspension using cell growth medium (cell density approximately 1~2×10⁻⁶). 5 (cells / mL) 3.4.3 Cell plating: Add 0.1 mL of cell suspension to each well of a 96-well plate and incubate at 37°C and 5% CO2 for about 24 h (to achieve a cell growth density of about 80% to 90% per well). 3.4.4 Drug preparation: Weigh an appropriate amount of drug and prepare a drug stock solution with a concentration of 3.52 nmol / μL according to the ratio of drug mass (mg): 80% ethanol volume (mL) = 21:13. Then prepare a drug solution with a concentration of 179.0085 nmol / L according to the method in the table and filter it through a 0.45 μm sterile filter. Table 9 3.4.5 Drug dilution: Based on experimental requirements, assess and select appropriate dilution ratios (such as 3x, 4x, 5x, 6x, and 7x) and dilute the drug solution to eight concentrations using virus diluent.

[0067] 3.4.6 Virus dilution: The TCID of each virus suspension sample was measured according to step 3.3. 50 Take a sufficient amount of the harvested virus suspension from each sample, mix well, and dilute each virus suspension to the appropriate multiple (100 TCID50) using virus diluent. 50 / 0.05 mL-400 TCID 50 / 0.05 mL); 3.4.7 96-well plate setup: Take a new 96-well plate (plate A) and set up cell control, test samples (maximum 2 cases), reference virus strain, and virus control (each test sample and reference virus strain has its corresponding virus control). The cell control has 12 wells, the test samples and reference virus strains each have 3 wells, and the test samples and reference virus strains each have 4 wells. 3.4.8 Virus drug incubation: Add 80 μL of the virus solution corresponding to the test sample and 80 μL of virus diluent to the virus control wells, respectively. Add 80 μL of the virus solution corresponding to the test sample and 80 μL of the drug solution of the corresponding concentration to the test wells, respectively. Add 160 μL of virus diluent to the cell control wells, mix well, and incubate at 37°C for 2 h. 3.4.9 Virus inoculation: After incubation, wash the 96-well cell culture plate (B plate) with a cell density of 80%-90% with PBS. Transfer 100 μL of solution from each well of plate A to the corresponding well of plate B and incubate at 35°C and 5% CO2 for 48 h. 3.4.10 Preparation of CCK-8 working solution: CCK-8 working solution is prepared by mixing CCK8 solution and MDCK cell growth medium at a ratio of CCK8 solution:MDCK cell growth medium = 1:10; 3.4.11 After the B-plate virus inoculation culture is completed, add 150 μL PBS to each well and wash the cell plate twice. Aspirate the PBS, add 110 μL CCK-8 working solution to each well, and continue to incubate at 37℃ and 5% CO2 for 1~2 h. 3.4.12 Plate Reading: After the culture was completed, the absorbance of each well was measured using a microplate reader with a main wavelength of 450 nm and a reference wavelength of 620 nm, and the IC50 of each sample was calculated using GraphPad Prism 8. 50 ; Table 10 3.4.13 Virus inhibition rate = (OD value of experimental wells - mean OD value of virus control wells) / (mean OD value of cell control wells - mean OD value of virus control wells).

[0068] Table 11 4. Interpretation of Results 4.1 When the virus to be detected is IC 50 Value <10 times that of reference strain IC 50 A value of 1 indicates that the virus is sensitive to ZSP1273; 4.2 When the reference strain IC is 10 times 50 Value ≤ Detected virus IC 50 Value ≤ 100 times the reference strain IC 50 A value of 1 indicates that the virus's sensitivity to ZSP1273 is reduced; 4.3 When the virus to be detected is IC 50 Value >100 times that of the reference strain IC 50 A value of 1 indicates that the virus's sensitivity to ZSP1273 is highly reduced; Note: If the influenza A virus to be tested is IC 50 Value ≥ 10 times the reference strain IC 50 When the value is reached, the virus requires three independent tests. If the average of the three test results is still ≥10 times the reference strain IC50 value, the test is considered successful. 50 If the value is not specified, it will be interpreted according to the corresponding standard (sensitivity is reduced).

[0069] Experimental results: Test sample Z1 IC 50 Value (0.3516) < 10 times the IC50 of the reference virus strain 50 The value (0.3487) indicates sensitivity to the drug. The results are shown in Table 12.

[0070] Table 12 Example 3 Method validation experiment.

[0071] To verify the accuracy and repeatability of the detection method of the present invention, this embodiment also uses conventional detection methods in the prior art for parallel determination as a control example. The results are shown in Tables 13 and 14. The accuracy and repeatability of the method of the present invention have been verified, demonstrating good accuracy and repeatability.

[0072] Table 13 Table 14 In summary, the method of the present invention effectively solves the problems of existing technologies, such as failure to reflect actual drug efficacy, narrow applicability, and strong subjectivity.

[0073] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for analyzing influenza virus drug resistance, characterized in that, The method for analyzing influenza virus drug resistance includes: co-incubating an anti-influenza drug with an influenza virus to obtain a drug-virus incubation mixture; inoculating the drug-virus incubation mixture into host cells; culturing the inoculated host cells to allow the virus to infect the cells and replicate; mixing the cultured cells with a cell proliferation activity detection reagent; assessing the degree of inhibition of viral replication by the drug based on cell proliferation activity; and determining the drug resistance of the influenza virus.

2. The method for analyzing influenza virus drug resistance according to claim 1, characterized in that, Influenza viruses include any one or a combination of at least two of the following: influenza A virus, influenza B virus, influenza C virus, or influenza D virus.

3. The method for analyzing influenza virus drug resistance according to claim 1 or 2, characterized in that, The host cells include MDCK cells and / or MDCK-STX cells.

4. The method for analyzing influenza virus drug resistance according to any one of claims 1-3, characterized in that, The co-incubation temperature is 35-38℃, and the time is 1-3 hours; Preferably, the culture temperature is 30-40℃ and the culture time is 36-60 h.

5. The method for analyzing influenza virus drug resistance according to any one of claims 1-4, characterized in that, The cell proliferation activity assay reagent includes the CCK-8 reagent.

6. The method for analyzing influenza virus drug resistance according to any one of claims 1-5, characterized in that, The evaluation of the drug's inhibitory effect on viral replication includes: calculating the viral inhibition rate, and calculating the drug's half-maximal inhibitory concentration (IC50) based on the viral inhibition rate. 50 ; The formula for calculating the virus inhibition rate is shown in equation (1); Inhibition rate = (OD value of experimental wells - mean OD value of virus control wells) / (mean OD value of cell control wells - mean OD value of virus control wells) Equation (1).

7. The method for analyzing influenza virus drug resistance according to any one of claims 1-6, characterized in that, The determination of influenza virus drug resistance includes: setting the half-maximal inhibitory concentration (IC50) of the virus to be tested. 50 Value and reference virus half-maximal inhibitory concentration (IC50) 50 The values ​​are compared, and the judgment criteria are as follows: When the half-inhibitory concentration (IC50) of the virus to be detected 50 Value < 10 times the reference plant's half-maximal inhibitory concentration (IC50) 50 When the value is [value missing], it is determined to be sensitive to the drug; When the half-maximal inhibitory concentration (IC50) of the reference plant was 10 times that of the reference plant... 50 Value ≤ IC50 of the virus to be detected 50 Value ≤ 100 times the reference plant's half-maximal inhibitory concentration (IC50) 50 When the value is [value missing], it is determined to be a decrease in drug sensitivity; When the half-inhibitory concentration (IC50) of the virus to be detected 50 Value > 100 times the reference plant's half-maximal inhibitory concentration (IC50) 50 When the value is [value missing], it is judged as a highly reduced sensitivity to the drug; Preferably, when the half-maximal inhibitory concentration (IC50) of the virus to be detected is... 50 Value ≥ 10 times the reference plant's half-maximal inhibitory concentration (IC50) 50 When the value is reached, at least three independent repeated tests should be performed, and the average value should be used as the final judgment basis.

8. The method for analyzing influenza virus drug resistance according to any one of claims 1-7, characterized in that, The method for analyzing influenza virus drug resistance also includes: performing a half-maximum tissue culture infectious dose (TCID50) test on the virus sample before co-incubating the anti-influenza virus with the antiviral drug to determine the viral infection titer; Preferably, the half-maximal tissue culture infection dose (TCID50) is calculated using the Reed-Muench method; Preferably, the anti-influenza virus drug comprises any one or a combination of at least two of the following: neuraminidase inhibitors, PA protein inhibitors, or PB2 subunit inhibitors of RNA polymerase.

9. The use of the influenza virus drug resistance analysis method according to any one of claims 1-8 in the preparation of products for clinical efficacy evaluation of anti-influenza virus drugs.

10. The application according to claim 9, characterized in that, The influenza virus includes any one or a combination of at least two of the following: influenza A virus, influenza B virus, influenza C virus, or influenza D virus.

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