Evaluation method for virus killing effect of virus aerosol

Abstract

The invention relates to the technical field of disinfection effect evaluation, and discloses a method for evaluating the virus disinfection effect of virus aerosol. According to the method for evaluating the virus killing effect of the virus aerosol, a real virus is simulated by adopting a pseudovirus, so that the biological safety risk is avoided; through the innovative pseudovirus activity detection method, the experimental operation complexity is greatly reduced, the detection time is shortened, meanwhile, the detection sensitivity is improved, and the method can be applied to the disinfection effect test and evaluation of scientific research and commercial air disinfection equipment (such as ultraviolet disinfection equipment).

Description

Technical Field

[0001] The present invention relates to the technical field of disinfection effect evaluation, and in particular to a method for evaluating the virus disinfection effect of virus aerosols. Background Art

[0002] In the disinfection effect evaluation technology system, the killing rate of indicator microorganisms is generally used to evaluate the disinfection effect. For example, in the "Disinfection Technical Specifications" (2002), Staphylococcus aureus is used as an indicator microorganism when evaluating the disinfection effect of air, and polio virus type I vaccine strain is used as an indicator microorganism when evaluating the disinfection effect of viruses; in the "Ultraviolet Disinfector Hygiene Requirements", artificial spray Staphylococcus aureus aerosol is used to evaluate the minimum effective dose of air disinfection of ultraviolet air disinfectors. It can be seen that in the existing disinfection effect evaluation system, bacteria are usually used as indicator microorganisms.

[0003] However, some disinfection methods have different effects on bacteria and viruses. For example, when ultraviolet rays are used for disinfection, viruses are significantly less sensitive to ultraviolet rays than bacteria. Using bacteria alone as indicator microorganisms is obviously not enough to indicate all pathogenic microorganisms in the air. Related technologies also propose to use real viruses in the laboratory to conduct evaluation experiments on the disinfection effect of viruses, but these methods are limited to the laboratory and face extremely high biosafety risks. In addition, the experimental steps of virus activity detection are many and the operation is complicated, which is not conducive to promotion and application.

[0004] Therefore, it is difficult to accurately, safely and easily evaluate the virus disinfection effect in the existing disinfection effect evaluation system. Summary of the invention

[0005] In view of this, the present invention provides a method for evaluating the virus disinfection effect of virus aerosols to solve the problem that it is difficult to accurately, safely and simply evaluate the virus disinfection effect in the existing disinfection effect evaluation system.

[0006] In a first aspect, the present invention provides a method for evaluating the virus disinfection effect of virus aerosols, comprising the following steps: The pseudovirus aerosol generating liquid is used to generate pseudovirus aerosol in the experimental chamber, wherein the pseudovirus aerosol generating liquid contains pseudovirus, the pseudovirus has host cell infection ability and carries a fluorescent protein gene; Performing virus disinfection treatment on the pseudovirus aerosol, and collecting the aerosol after virus disinfection treatment to obtain a pseudovirus aerosol collection liquid; Using the pseudovirus aerosol collection solution to infect host cells, and detecting the number of host cells expressing fluorescent proteins after the infection is completed; Based on the number of host cells expressing the fluorescent protein, the virus killing rate of the virus disinfection treatment for viral aerosols is determined.

[0007] In the above-mentioned evaluation method for the virus disinfection effect of viral aerosols, biosafety risks are avoided by using pseudoviruses to simulate real viruses; through innovative pseudovirus activity detection methods, the complexity of experimental operations is greatly reduced, the detection time is shortened, and the sensitivity of detection is increased. It can be applied to scientific research and commercial air disinfection equipment (such as ultraviolet disinfection equipment) disinfection effect testing and evaluation.

[0008] Specifically, by adopting the above evaluation method, the present invention has at least the following beneficial effects: (1) The present invention uses pseudoviruses to evaluate the disinfection effect of viral aerosols. After the virus infects the host cells, it only needs to be cultured for one day (24 hours) to be detected, which is 2-3 days shorter than similar technologies. (2) The pseudovirus used in the present invention carries a fluorescent protein gene. After the pseudovirus infects the host cell, the host cell can express the fluorescent protein. The cell count can be directly performed using a fluorescent cell counter. The traditional qPCR method for detecting the infectivity titer of the pseudovirus requires nucleic acid extraction, reverse transcription, and PCR amplification. Therefore, the present invention is simpler and faster in experimental operation; (3) The present invention uses pseudoviruses to simulate real viruses, avoiding high biosafety risks and having a wider range of application scenarios; (4) The present invention transforms the observation of infectious viruses into imaging of cells expressing fluorescence, making the detection results more realistic and intuitive; (5) The present invention completely simulates the situation in which real viruses form viral aerosols in the air, overcoming the technical difficulties of producing a large amount of infectious viral aerosols when using real viruses in related technologies and the low detection sensitivity of real active viruses, making it possible to directly use fake viruses to carry out evaluation experiments on the disinfection effect of viral aerosols.

[0009] In an optional embodiment, the pseudovirus is obtained by packaging a pseudovirus vector into a cell plasmid, wherein the cell plasmid includes a fluorescent protein gene transfer plasmid, and the fluorescent protein gene is inserted into the fluorescent protein gene transfer plasmid; Optionally, the pseudovirus vector includes but is not limited to a lentivirus vector, an adenovirus vector and a VSV virus vector; Optionally, the cell plasmid further includes an envelope plasmid and / or a backbone plasmid.

[0010] In an optional embodiment, the preparation process of the pseudovirus includes: packaging cell plasmids with pseudovirus vectors, then transfecting into pseudovirus packaging cells, culturing for 48 to 72 hours, obtaining cell lysate or cell culture medium, and obtaining a pseudovirus solution containing the pseudovirus.

[0011] In an optional embodiment, the pseudovirus aerosol generating liquid is obtained by concentrating the pseudovirus solution, and the concentration method may include: Add 0.2 mol / L to 5 mol / L NaCl and 1% to 20% PEG8000 (W / V) to the pseudovirus solution obtained by culture, and then place it in a refrigerator at 4°C overnight, invert and mix 3 to 4 times, and then centrifuge at 3000g to 10000g at 4°C for 10 to 90 minutes. Discard the supernatant and dissolve the precipitate with 1×PBS.

[0012] In an optional embodiment, the host cells include but are not limited to HEK293T cells, HEK293 cells and Jurkat cells. When the pseudovirus aerosol collection solution is used to infect the host cells, the infection time is 18 to 72 hours.

[0013] In an optional embodiment, the fluorescent protein gene includes but is not limited to a green fluorescent protein gene, a red fluorescent protein gene and a yellow fluorescent protein gene.

[0014] In an optional embodiment, before the pseudovirus aerosol generating liquid generates pseudovirus aerosol in the experimental chamber, the method further comprises: Conducting an infectivity titer test on the pseudovirus aerosol generating liquid to obtain an actual infectivity titer of the pseudovirus aerosol generating liquid; When the actual infectivity titer is lower than the preset infectivity titer, the pseudovirus aerosol generating liquid is re-prepared.

[0015] In an optional embodiment, the preset infectivity titer is 5×10 5 IU / mL~5×10 8 IU / mL.

[0016] In an optional embodiment, the infectivity titer of the pseudovirus aerosol generating liquid can be detected by the following method: Take the host cells, inoculate them into the cell culture plate according to the appropriate number of cells per well and culture medium volume, and place them in the cell culture incubator for culture; take the pseudovirus aerosol generator and dilute it in a gradient manner using the cell culture medium (dilution ratio: k); before infection, take the cells on the cell culture plate and count them to obtain the average number of cells per well before infection (m, unit: cells); take out the cells cultured overnight, discard all the cell culture medium, add the dilution of the virus aerosol generator for infection, and return to the cell culture incubator for culture for at least 24 hours; take out the infected cells, disperse the cells into single cells, and transfer them to a centrifuge tube; centrifuge the cells at an appropriate speed and time; discard the supernatant and resuspend the cells with PBS; use a fluorescent cell counter to count the cells expressing fluorescence to obtain the ratio of the number of cells expressing fluorescence to the total number of cells (infection ratio or transduction efficiency: f); according to the dilution ratio, calculate the infectious virus titer of the virus aerosol generator at this volume (t, unit: IU, IU: Infectous units):

[0017] The standard curve was prepared using the dilution gradient of the virus aerosol generating liquid and the corresponding virus titer to obtain the linear equation of the standard working curve (within the linear range). The infectious virus titer (T, unit: IU / mL) of the virus aerosol generating liquid per unit volume was then calculated based on the linear equation of the standard curve.

[0018] In an optional embodiment, aerosols are collected using a virus aerosol sampling solution, wherein the virus aerosol sampling solution includes a cell culture medium; Optionally, the amount of the virus aerosol sampling solution used is 4 mL to 20 mL; Optionally, the cell culture medium includes DMEM medium and / or 1640 medium; Optionally, the cell culture medium contains 0% to 20% fetal bovine serum.

[0019] In an optional embodiment, when the virus aerosol sampling liquid is used to collect aerosols, the virus aerosol sampling liquid is placed in a planktonic bacteria sampler, and the aerosols are collected according to a preset sampling flow rate and sampling time. Optionally, the sampling flow rate of the planktonic bacteria sampler is 28.3 L / min to 100 L / min, and the sampling time is 1 min to 10 min.

[0020] In an optional embodiment, when the pseudovirus aerosol generating liquid generates pseudovirus aerosol in the experimental chamber, the pseudovirus aerosol is generated under a preset pressure, temperature and humidity. Optionally, the pressure in the experimental chamber is controlled to be -1 Pa to -30 Pa, the temperature is 21°C to 29°C, and the humidity is 40% to 70%.

[0021] In an optional embodiment, determining the virus disinfecting rate of the virus disinfection treatment for virus aerosols based on the number of host cells expressing the fluorescent protein includes: Based on the number of host cells expressing fluorescent protein in the experimental group and the number of host cells expressing fluorescent protein in the control group, determining the virus killing rate of the virus disinfection treatment for the viral aerosol; Among them, the pseudovirus aerosol in the experimental group was disinfected before the pseudovirus aerosol collection fluid was collected, while the pseudovirus aerosol in the control group was not disinfected before the pseudovirus aerosol collection fluid was collected.

[0022] In an optional embodiment, after the infection is completed, the number of host cells expressing the fluorescent protein can be detected by the following method: Take out the pseudovirus aerosol collection solution, add it to the pre-cultured host cells, and infect for at least 24 hours; take out the cells and disperse them into single cells; transfer the cells to a centrifuge tube and centrifuge the cells at an appropriate speed and time; aspirate the supernatant, add PBS to resuspend the dispersed cells; take an appropriate amount of PBS-resuspended cell suspension and add it to the counting slot of the cell counting plate, and insert the cell counting plate into the cell counter; select the fluorescence imaging mode, perform fluorescence imaging on the sample to be tested, and the cell counter automatically identifies the fluorescence imaging photos and gives the number of cells expressing fluorescent protein in the image. The number of cells expressing fluorescent protein is positively correlated with the number of viruses with infectious activity. When the proportion of fluorescent cells to the total number of cells is less than 10%, the two numbers can be considered equal; count the number of active viruses detected in the experimental group (Nt, unit: individual) and the number of active viruses detected in the control group (Nc, unit: individual), and calculate the killing rate (K) of the relevant method or equipment for the virus aerosol:

[0023] In an optional embodiment, the present invention provides a method for evaluating the virus disinfecting performance of an ultraviolet disinfection device based on pseudovirus aerosol, comprising the following steps: (1) Packaging and preparing pseudoviruses that can infect host cells and carry fluorescent protein genes, and concentrating the pseudoviruses to serve as pseudovirus aerosol generating liquid; (2) Conduct infectious titer testing on the pseudovirus aerosol generating fluid; (3) Add the pseudovirus aerosol generating liquid into the aerosol generator, hang it in the negative pressure aerosol test chamber, and arrange two ultraviolet air disinfection devices for the experimental group and the control group; (4) Pseudovirus aerosols occurred in the negative pressure aerosol test chamber; (5) Turn on the ultraviolet air disinfection equipment in the experimental group and start the floating bacteria sampler at the same time to obtain the pseudovirus aerosol collection liquid; (6) Use pseudovirus aerosol collection fluid to infect host cells, use a fluorescent cell counter to detect the number of infected host cells expressing fluorescent protein, and calculate the killing rate of virus aerosol by ultraviolet air disinfection equipment based on the data of the experimental group and the control group.

[0024] Optionally, the arrangement of the negative pressure aerosol test chamber in the above step (3) is as follows: Hang the aerosol generator on the upper part of the negative pressure aerosol experimental cabin, and insert the air compressor outlet pipe into the air inlet of the aerosol generator; place two identical ultraviolet air disinfection devices in the negative pressure aerosol experimental cabin, and the two devices are symmetrically distributed on both sides of the generator, and the air inlets of the two ultraviolet air disinfection devices are opposite to the air outlets of the aerosol generator; add the virus aerosol sampling liquid into the culture dish, and place the culture dish into the sampling head of the planktonic bacteria sampler; place two planktonic bacteria samplers with the same settings into the aerosol experimental cabin, and the sampling heads of the planktonic bacteria samplers are respectively connected to the air outlets of the two ultraviolet air disinfection devices, and sealed; close the aerosol experimental cabin door, turn on the control and monitoring equipment of the pressure, temperature and humidity in the cabin, and adjust the pressure, temperature and humidity parameters to reach the specified range.

[0025] Preferably, the aerosol generator in step (3) is a Collison bioaerosol generator, and the effect of generating aerosol is not affected by the bubbles generated by the aerosol generating liquid. The negative pressure aerosol test chamber has a volume of 1.5 to 20 m 3 The enclosed space is connected to the air compressor and the vacuum pump through pipes to control the internal air pressure, temperature and humidity. At the same time, pressure, temperature and humidity sensors are installed to monitor the internal air pressure, temperature and humidity parameters in real time through a computer.

[0026] Preferably, an ultraviolet lamp and a fan are installed inside the ultraviolet air disinfection equipment. Under the action of the fan, external air enters from one end of the box, passes through the ultraviolet lamp irradiation area inside the box, and then goes out through the opening at the other end, thereby completing the disinfection of the air.

[0027] Preferably, the air sampler is an impact-type planktonic bacteria sampler, and its working principle is as follows: the air inlet head has hundreds of air inlet holes with a diameter of 0.3 mm, which are arranged in a circle with a diameter not exceeding 10 cm. A culture dish containing air sampling liquid is placed under the air inlet holes. When the sampler is started, negative pressure is formed inside due to air extraction, and the external virus aerosol quickly enters the air inlet holes together with the air, and impacts the air sampling liquid, and the virus aerosol will be captured by the air sampling liquid. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0029] Figure 1 This is a cell fluorescence imaging recognition result diagram of Example 2 of the present invention; Figure 2 is a standard curve diagram of Example 2 of the present invention; Figure 3 is a specific layout diagram of the negative pressure aerosol experimental chamber of Example 3 of the present invention; Figure 4 This is a diagram of cell fluorescence imaging identification results for the experimental group and the control group in Example 3 of the present invention. DETAILED DESCRIPTION

[0030] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0031] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0032] The present invention is further described in detail below in conjunction with specific embodiments. These embodiments should not be construed as limiting the scope of protection claimed by the present invention. Example 1

[0033] This example is used to illustrate the preparation method of pseudovirus aerosol generating liquid: (1) Amplification of green fluorescent protein gene using PCR technology egfp , using molecular cloning technology to clone the green fluorescent protein gene egfp Transfer plasmids inserted into the lentiviral packaging system ikB Multiple cloning site region to obtain the transfer plasmid of the new lentiviral vector packaging system pCDH-egfp ; (2) The prepared lentiviral vector was used to package the cell plasmid, and the obtained product was transfected into HEK293T cells using lipo3000 transfection reagent, and cultured for 48 hours after the transfection. Among them, the cell plasmid includes pMD2.G(Envelope plasmid), pCMV-dR8.91 (backbone plasmid) and pCDH-egfp (transfer plasmid); (3) After the culture is completed, the cell culture medium is transferred to a 50 mL centrifuge tube and centrifuged at 4000 g / min at 4°C for 5 min. After the centrifugation is completed, the supernatant is transferred to a clean 50 mL centrifuge tube to obtain the pseudovirus solution; (4) Add PEG8000 (final concentration of 10% (w / v)) and NaCl (final concentration of 1 mol / L) to the pseudovirus solution obtained in step (3), mix by inversion, and place in a refrigerator at 4°C overnight (more than 12 hours), inverting and mixing several times during the process; the resulting product is centrifuged at 4000 g / min at 4°C for 30 min. After centrifugation, the supernatant is discarded, and 1×PBS (the added volume is 1 / 10 of the volume of the pseudovirus solution) is added to the resulting precipitate. Use a micropipette to gently pipette to redissolve the precipitate to obtain a concentrated pseudovirus solution, which is used as a pseudovirus aerosol generating solution. Example 2

[0034] This example is used to illustrate the detection method of the infectivity titer of the pseudovirus aerosol generating liquid: (1) HEK293T cells were inoculated into 24-well plates for culture, with 4×10 cells added to each well. 5 The volume of the culture medium (DMEM high glucose, containing 10% fetal bovine serum and 1% penicillin-streptomycin double antibody) was 500 μL; (2) The next day, cells from some wells of the 24-well plate were taken and counted using a cell counter. The results are shown in Table 1. Based on Table 1, the average number of cells per well before infection was 661,333; Table 1 Count of cells in the wells before infection

[0035] (3) Take the pseudovirus aerosol generating solution and dilute it 10 times, and then perform gradient dilution of the diluted virus solution: take 5 1.5mL centrifuge tubes, add 200, 220, 240, 260, and 280μL of DMEM culture medium containing 2% FBS, and then add 100, 80, 60, 40, and 20μL of virus dilution solution respectively to obtain the virus gradient dilution solution used to infect cells, with a volume of 300μL; (4) Take the cells from the 24-well plate, remove all the cell culture medium in the wells, add the virus gradient dilution solution, and continue to culture in a 37°C incubator for 24 hours; (5) After the culture is completed, remove the cells from the 24-well plate, remove all the virus gradient dilutions, add 200 μL of 0.25% trypsin solution and digest at 37°C for 2 min, then add 800 μL of DMEM complete medium to terminate the digestion, blow off the cells and transfer them to a 1.5 mL centrifuge tube, and centrifuge at 3000 rpm for 3 min; (6) Take the centrifuged cells, remove the supernatant, add 180 μL 1× PBS, and centrifuge at 3000 rpm for 2 min; take the centrifuged cells, remove the supernatant, and add 100 μL 1× PBS to resuspend the dispersed cells; (7) Take the resuspended and dispersed cells and add them to the counting slot of the cell counting plate. Put the cell counting plate into the cell counter to perform fluorescent imaging and automatic identification of the cells. The identification results are as follows: Figure 1 As shown; then the ratio of the number of fluorescent cells is given, and the infectious titer of each virus gradient dilution is calculated using the ratio of fluorescent cells. The results are shown in Table 2; (8) Use the infectious titer of the virus gradient dilution to draw a standard curve, such as Figure 2 As shown; Based on the standard curve, the infectious titer of each milliliter of pseudovirus aerosol generating liquid was calculated. According to the standard curve formula, the infectious titer per unit volume of the virus aerosol generating liquid was calculated to be: 1.06×10 7 IU / mL.

[0036] Table 2 Infectious titer of each virus gradient dilution Example 3

[0037] The disinfection effect of a certain brand of ultraviolet air disinfection machine was evaluated and tested, including the generation, disinfection and collection of packaged virus aerosols, as well as the detection of virus aerosol collection fluids collected from the experimental group and the control group, and finally the calculation of the killing rate: (1) 20 mL of the pseudovirus aerosol generating liquid prepared in Example 1 was added to the negative pressure aerosol generator, and then the aerosol generator was placed in the upper part of the aerosol test chamber. The aerosol test chamber was 1.5 m long, 1 m wide and 1 m high, with a total volume of 1.5 m 3 , the air outlet of the aerosol generator is 80 cm away from the bottom of the aerosol test chamber, 75 cm away from the left wall, and 20 cm away from the inner wall. Insert the compressor ventilation pipe into the air inlet of the generator; (2) Place two ultraviolet air disinfection devices in the aerosol test chamber, with the air inlet of the device facing the air outlet of the aerosol generator. The two devices are symmetrically distributed on both sides of the generator, and the air outlet is at the same height as the air outlet of the aerosol generator. The air inlets of the two disinfection devices are 20 cm apart, and the midpoint of the air inlet line is 20 cm away from the aerosol generator; (3) Add 6 mL of virus aerosol sampling solution (DMEM culture medium containing 2% FBS) into the culture dish, and install the culture dish in the airborne bacteria sampler; place two airborne bacteria samplers with the same settings into the aerosol experimental chamber, with a sampling flow rate of 100 L / min and a sampling time of 10 min. The air inlet of the sampler is airtightly connected to the air outlet of the two ultraviolet air disinfection devices; (4) After steps (1) to (3), the specific layout of the negative pressure aerosol test chamber is as follows: Figure 3 ; Close the aerosol experiment cabin, turn on the control and monitoring equipment for pressure, temperature and humidity in the cabin, and adjust the pressure to -10 Pa, the temperature to 22-25℃ and the humidity to 50-70%; (5) Starting the compressor connected to the virus aerosol generator to start generating pseudo-virus aerosol; (6) Turn on the ultraviolet air disinfection equipment. The two ultraviolet air disinfection equipment are set as a control group and an experimental group. The control group equipment does not turn on ultraviolet irradiation, and the other settings are the same as the experimental group; (7) Start the floating bacteria sampler and run it according to the preset sampling flow and sampling time; (8) After the sampling of the floating bacteria sampler is completed, the generation of pseudovirus aerosols is stopped and the ultraviolet air disinfection equipment is turned off; (9) Open the aerosol test chamber, take out the floating bacteria sampler, and take out the pseudovirus aerosol sampling liquid; (10) One day before the experiment, HEK293T cells were seeded into a 24-well plate, with 4 × 10 cells per well. 5 The volume of culture medium (DMEM high glucose, containing 10% fetal bovine serum and 1% penicillin-streptomycin double antibody) in each well was 500 μL; after removing the pseudovirus aerosol sampling liquid, all the culture medium inoculated with HEK293T cells in the 24-well plate was aspirated, 1 mL of pseudovirus aerosol sampling liquid was added, and then the culture plate was returned to the cell culture incubator at 37°C for infection for 24 hours; (11) After infection, remove the cell culture plate, remove all the culture medium, add 200 μL of 0.25% trypsin solution, digest the cells at 37°C for 2 min, then add 800 μL of DMEM complete medium to terminate digestion, while pipetting to disperse the cells; (12) Transfer the dispersed cells to a 1.5 mL centrifuge tube and centrifuge at 3000 rpm for 3 min. After removing the supernatant, add 180 μL of 1× PBS solution and centrifuge at 3000 rpm for 2 min. After removing the supernatant, add 100 μL of 1× PBS solution and pipette to evenly disperse the cells in the solution. (13) Take 20 μL of the cell solution obtained in step (12) and add it to the counting slot of the cell counter counting plate. Insert the counting plate into the cell counter and perform fluorescence imaging of the cells using the fluorescence imaging mode. Figure 4 These are representative pictures of the experimental group and the control group. Based on the counts of fluorescent cells in the experimental group and the control group using a cell counter, the disinfection effect of the ultraviolet air disinfector was calculated, and the results are shown in Table 3.

[0038] Table 3 Virus disinfection effect of a certain brand of ultraviolet air disinfection machine

[0039] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for evaluating the virus disinfection effect of virus aerosols, characterized in that: The steps include: The pseudovirus aerosol generating liquid is used to generate pseudovirus aerosol in the experimental chamber, wherein the pseudovirus aerosol generating liquid contains pseudovirus, the pseudovirus has host cell infection ability and carries a fluorescent protein gene; Performing virus disinfection treatment on the pseudovirus aerosol, and collecting the aerosol after virus disinfection treatment to obtain a pseudovirus aerosol collection liquid; Using the pseudovirus aerosol collection solution to infect host cells, and detecting the number of host cells expressing fluorescent proteins after the infection is completed; Based on the number of host cells expressing the fluorescent protein, the virus killing rate of the virus disinfection treatment for viral aerosols is determined.

2. The evaluation method according to claim 1, characterized in that: The pseudovirus is obtained by packaging a cell plasmid with a pseudovirus vector, wherein the cell plasmid includes a fluorescent protein gene transfer plasmid, and the fluorescent protein gene is inserted into the fluorescent protein gene transfer plasmid; Optionally, the pseudovirus vector includes but is not limited to a lentivirus vector, an adenovirus vector and a VSV virus vector; Optionally, the cell plasmid further includes an envelope plasmid and / or a backbone plasmid.

3. The evaluation method according to claim 1 or 2, characterized in that: The host cells include but are not limited to HEK293T cells, HEK293 cells and Jurkat cells.

4. The evaluation method according to any one of claims 1 to 3, characterized in that: The fluorescent protein gene includes but is not limited to a green fluorescent protein gene, a red fluorescent protein gene and a yellow fluorescent protein gene.

5. The evaluation method according to any one of claims 1 to 4, characterized in that: Before the pseudovirus aerosol generating liquid is made to generate pseudovirus aerosol in the experimental chamber, the method further comprises: Conducting an infectivity titer test on the pseudovirus aerosol generating liquid to obtain an actual infectivity titer of the pseudovirus aerosol generating liquid; When the actual infectivity titer is lower than the preset infectivity titer, the pseudovirus aerosol generating liquid is re-prepared.

6. The evaluation method according to claim 5, characterized in that: The preset infectious titer is 5×10 5 IU / mL~5×10 8 IU / mL.

7. The evaluation method according to any one of claims 1 to 6, characterized in that: Collecting aerosols using a virus aerosol sampling solution, wherein the virus aerosol sampling solution includes a cell culture medium; Optionally, the usage of the virus aerosol sampling solution is 4 mL to 20 mL; Optionally, the cell culture medium includes DMEM medium and / or 1640 medium; Optionally, the cell culture medium contains 0% to 20% fetal bovine serum.

8. The evaluation method according to claim 7, characterized in that: When the virus aerosol sampling liquid is used to collect aerosols, the virus aerosol sampling liquid is placed in a planktonic bacteria sampler, and the aerosols are collected according to a preset sampling flow rate and sampling time.

9. The evaluation method according to any one of claims 1 to 8, characterized in that: When the pseudovirus aerosol generating liquid is made to generate pseudovirus aerosol in the experimental chamber, the pseudovirus aerosol is generated under a preset pressure, temperature and humidity.

10. The evaluation method according to any one of claims 1 to 9, characterized in that: The method of determining the virus disinfecting rate of the virus disinfection treatment for the virus aerosol based on the number of host cells expressing the fluorescent protein comprises: Based on the number of host cells expressing fluorescent protein in the experimental group and the number of host cells expressing fluorescent protein in the control group, determining the virus killing rate of the virus disinfection treatment for the viral aerosol; Among them, the pseudovirus aerosol in the experimental group was disinfected before the pseudovirus aerosol collection fluid was collected, while the pseudovirus aerosol in the control group was not disinfected before the pseudovirus aerosol collection fluid was collected.

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