CN113416711A - SARS-COV-2 virus strain, mouse model construction method and application - Google Patents
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Abstract
The invention provides a SARS-COV-2 virus strain, a mouse model construction method and application, belonging to the technical field of animal model construction. The SARS-COV-2 virus strain has the preservation number of CCTCCNO: V202138. The invention provides a method for constructing a mouse model infected by SARS-COV-2 virus, which comprises the steps of anesthetizing an hACE2 mouse, and infecting the anesthetized hACE2 mouse with a virus liquid containing the SARS-COV-2 virus strain. The mortality rate of the constructed mouse model is up to 95.83% +/-7.22% in 5 days after infection, the clinical symptoms of the mouse model approach the clinical reaction of human, and meanwhile, in the research of vaccines and antiviral drugs, the detection result of the lung tissue live virus titer of the mouse model can reflect the real drug effect condition, so the mouse model obtained by the construction method provided by the invention can be widely applied to the screening of the new corona antiviral drugs and vaccines in the preclinical research.
Description
Technical Field
The invention belongs to the technical field of construction methods of animal models, and particularly relates to a SARS-COV-2 virus strain, a construction method of a mouse model and application of the mouse model.
Background
SARS-COV-2 virus is a pathogenic virus of severe acute respiratory syndrome and has strong infectivity. The two main methods for resisting SARS-COV-2 virus are vaccination and the use of antiviral drugs. At present, specific drugs against SARS-COV-2 virus are not available.
Screening specific medicine for resisting SARS-COV-2 virus can not be separated from animal model of disease. The existing SARS-COV-2 virus pneumonia animal models comprise a human ACE2 transgenic mouse model, a non-transgenic novel coronavirus pneumonia mouse animal model, a rhesus new coronary pneumonia model, a model of new coronary virus infection ferret, hamster and the like. Among the above animal models, the relationship between the nonhuman primate rhesus monkey and human is the closest in terms of evolutionary relationship, and is theoretically the most ideal experimental animal model. However, the experimental monkey has limited source, high experimental related cost, large animal biosafety third-level laboratory and limited experimental site, so the application of the model in the evaluation of anti-inflammatory drugs is greatly limited. Ferrets and hamsters are also limited in animal origin and quantity. And the management cost of the mouse as an experimental animal is low, and the mouse is often the first choice of an animal model under the condition of similar other conditions.
There are two current mouse models of coronary pneumonia: one was a mouse model transduced with a viral vector carrying the human ACE2 receptor, and the other was a mouse model transgenic for hACE 2. The virus vector transduction mouse model directly introduces a novel coronavirus receptor into the lung of a mouse by a receptor vector transduction technology, so that the mouse is susceptible to viruses. The mice do not need special reproduction and are suitable for large-scale popularization in a short period, but the mice model infected by the new coronavirus only has an excessive weight reducing symptom and can not cause death. Therefore, the model is probably not the best model which can truly reflect the pathogenicity of the new corona virus, and certain limitation is brought to the application of the model in an evaluation system for evaluating the anti-new corona medicine.
It has been reported in the prior art that a new coronavirus mouse model was constructed by infecting a hACE2 transgenic mouse with the SARS-COV-2 strain, but the constructed mouse model showed no mild sign of infection until day 4 weight loss, but no other obvious clinical symptoms were observed at day 5. Infected mice lost only about 10% of their body weight, but did not show distinct clinical symptoms. The pulmonary virus titer of all infected mice is very high, the hematology change is caused by pulmonary alveolar luminal protein fragments, interstitial inflammatory cell infiltration and pulmonary alveolar septal thickening, and the constructed mouse model cannot well simulate the clinical symptoms of human infection with the new coronavirus, so that great obstacle is brought to the screening of the anti-new coronavirus drugs.
Disclosure of Invention
In view of the above, the present invention aims to provide a SARS-COV-2 virus strain and its application, which is used to infect mouse to construct mouse model, and make the clinical symptoms of the constructed mouse model approach the clinical response of human.
The invention also aims to provide a method for constructing a mouse model infected by SARS-COV-2 virus, which has the characteristics of good safety and high success rate, and the constructed mouse model has good stability.
The invention provides a SARS-COV-2 virus strain, the preservation number of the SARS-COV-2 virus strain is CCTCC NO: V202138.
The invention provides application of the SARS-COV-2 virus strain in constructing a mouse model infected by SARS-COV-2 virus.
The invention provides a method for constructing a mouse model infected by SARS-COV-2 virus, which comprises the following steps:
after anesthetizing hACE2 mice, the anesthetized hACE2 mice were infected with a virus fluid containing the SARS-COV-2 virus strain.
Preferably, the titer of said SARS-COV-2 virus strain in said virus liquid is not less than 107PFU/ml。
Preferably, the titer of the SARS-COV-2 virus strain is 104～106PFU/ml, the model is lethal, can be according to different experimental purpose selection virus titer.
Preferably, the mode of infection of hACE2 mice after anesthesia comprises a nasal drip mode of infection.
Preferably, the infection dose of the virus liquid is 45-55 mu L/virus.
The invention provides the application of the mouse model of SARS-COV-2 virus infection constructed by the method in screening the medicine for resisting SARS-COV-2 virus or the vaccine for preventing and controlling SARS-COV-2 virus.
The SARS-COV-2 virus strain provided by the invention has a preservation number of CCTCC NO: V202138. The virus strain can effectively replicate in the lung of a mouse, and a large amount of lymphocytes and monocytes in an alveolar stroma are infiltrated after 5 days of infection, and macrophages in an alveolar cavity are gathered, so that typical interstitial pneumonia and pathology are caused. The lung infected with the virus strain is matched with new coronary pneumonia in the aspects of pathological changes, body inflammatory factor storm, immune function disorder and the like, and can be used for constructing a new coronary virus infected mouse model.
Meanwhile, the virus strain of the invention has a virus attacking dose of 107PFU/mL, 5 days post infection lung titer 105.67PFU/mL, higher safety, and the prior art for the infection of the new coronavirusCompared with the challenge dose of a mouse model, the ideal challenge effect can be achieved under the condition of lower challenge dose and inoculation amount.
The invention provides a method for constructing a mouse model infected by SARS-COV-2 virus, which adopts the SARS-COV-2 virus strain to infect hACE2 transgenic mice, and the hACE2 transgenic mice infected by SARS-COV-2 virus show weight loss and death. The virus strain is rapidly replicated in the lung of an hACE2 transgenic mouse, and causes infiltration of a large number of lymphocytes and monocytes in an alveolar interstitium, and macrophage accumulation in the alveolar cavity, so that typical interstitial pneumonia and pathology are caused. The pathological changes of the lung, the body inflammatory factor storm and the immunologic dysfunction of the model are identical with the new coronary pneumonia, and the model can be used as an effective animal model for evaluating the anti-new coronary virus resistance of the anti-inflammatory drugs. Therefore, the novel coronavirus infection mouse model constructed by the method provided by the invention can simulate an animal model of severe human immunity characteristics caused by SARS-CoV-2 infection and can be used as a good model for evaluating the effectiveness of antiviral drugs or vaccines.
Meanwhile, the construction method provided by the invention has short modeling time, the weight of the mouse begins to lose and die after 48 hours of infection, the virus can be effectively replicated in the lung, the virus infection time can last for at least 5 days, and the mouse has continuous stability. The construction method can be repeated, and the success rate of modeling can reach 100%.
Drawings
FIG. 1 shows the body weight changes of mice infected with the new coronavirus K18hACE 2;
FIG. 2 is the results of the dissection of a mouse model after infection with SARS-CoV-2 new coronavirus; FIG. 2A: the fifth day of infection with SARS-CoV-2 New coronavirus showed a significant shrug, FIG. 2B: infection with SARS-CoV-2 New coronavirus developed lung and chest bloating on day five, FIG. 2C and FIG. 2D: emphysema appears on the fifth day after SARS-CoV-2 new coronavirus infection;
FIG. 3 shows the lung tissue inflammatory factor expression of mice of each group on the fifth day of infection with SARS-CoV-2 coronavirus;
FIG. 4 shows the pathological examination results (H-E,200X) of lung of mice of SARS-CoV-2 mice model group, wherein FIG. 4A shows moderate hemorrhage, severe interstitial pneumonia, moderate epithelial degeneration/necrosis and exfoliation of bronchioles and FIG. 4B shows moderate hemorrhage, moderate interstitial pneumonia, mild epithelial degeneration/necrosis and exfoliation of bronchioles and lung of mice of SARS-CoV-2 mice model group.
Biological material preservation information
The SARS-CoV-2 Virus strain (Novel Corona Virus) is preserved in the China center for type culture Collection at 24.5.24.2021, the preservation address is the Wuhan university school of eight No. 299 in the Wuchang district of Wuhan, Hubei province (the first attached small front of the Wuhan university), the preservation number of the Wuhan university is CCTCC NO: V202138, and the strain number is SARS-CoV-2/MO 15-1/B.
Detailed Description
The invention provides a SARS-COV-2 virus strain, the preservation number of the SARS-COV-2 virus strain is CCTCC NO: V202138.
In the present invention, the SARS-COV-2 virus strain is separated from the pharyngeal swab and the nasopharyngeal swab of the infected patient in the eighth national hospital of Guangzhou city, and the SARS-COV-2 new coronavirus is identified and confirmed. The identification method is preferably to detect SARS-COV-2 virus by a fluorescent RT-PCR method. The fluorescence RT-PCR method is used for detecting SARS-COV-2 virus, preferably for detecting Spike gene and ACE2 gene. The primers for RT-PCR detection of the Spike gene comprise forward primers and reverse primers shown in SEQ ID NO. 1 and SEQ ID NO. 2. The primers for the RT-PCR detection of the ACE2 gene comprise forward primers and reverse primers shown in SEQ ID NO. 3 and SEQ ID NO. 4. When the detection result is positive, the virus is SARS-COV-2 new coronavirus.
In the present invention, the method for culturing the SARS-COV-2 virus strain preferably comprises the following steps:
1) subculturing the Vero E6 cells 18-24 hours before inoculating the virus to obtain Vero E6 cells after subculture;
2) transferring virus liquid containing SARS-COV-2 virus strain into said passaged Vero E6 cell;
3) culturing Vero E6 cells inoculated with SARS-COV-2 strain, while shaking the culture container;
4) adding a new culture medium into the cultured Vero E6 cells, continuously culturing for 14-16 d, changing the culture medium every 3d, and collecting culture supernatant as virus liquid containing SARS-COV-2 virus strain.
In the present invention, the solvent of the virus solution in step 2) is preferably DMEM medium containing amphotericin B15 g/mL, penicillin 200IU/mL, and streptomycin 200 g/mL. The time for culturing in the step 3) is preferably 1-3 h, and more preferably 2 h. The time for shaking the culture container is preferably 12-17 min, and more preferably 15 min. The new culture medium in the step 4) is preferably a DMEM culture medium containing 2% of bovine serum, 15g/mL of amphotericin B, 200IU/mL of penicillin and 200g/mL of streptomycin by mass fraction. Continuously culturing until the cytopathic effect is achieved, and freezing and storing. The cytopathic effects include cell shrinkage, rounding, fusion, death and shedding. The cultivation process is preferably operated in a biosafety cabinet.
The invention provides application of the SARS-COV-2 virus strain in constructing a mouse model infected by SARS-COV-2 virus.
The invention provides a method for constructing a mouse model infected by SARS-COV-2 virus, which comprises the following steps:
after anesthetizing hACE2 mice, the anesthetized hACE2 mice were infected with a virus fluid containing the SARS-COV-2 virus strain.
In the present invention, the hACE2 mouse is preferably a commercial K18hACE2 mouse. The commercial K18hACE2 mouse is a transgenic mouse with human K18 gene and/or fragment as a promoter to drive hACE2 to be specifically expressed in epithelial cells. The source of the commercial K18hACE2 mouse is not particularly limited in the present invention, and a commercial K18hACE2 mouse known in the art may be used. In the present example, the commercial K18hACE2 mouse was purchased from Jiangsu Jiejicaokang Biotech, Inc. The age of the commercial K18hACE2 mouse is preferably 4 to 12 weeks, more preferably 5 to 10 weeks, and still more preferably 6 to 8 weeks. The commercial K18hACE2 mouse preferably has a body weight of 23 g/mouse to 35 g/mouse, more preferably 25 g/mouse to 32 g/mouse, and most preferably 28 g/mouse.
The method of anesthetizing mice according to the present invention is not particularly limited, and any method known in the art may be used. In the present example, the method of anaesthesia places mice in a desiccator with added isopfluorwan.
In the present invention, the titer of the SARS-COV-2 virus strain in the virus liquid is preferably not less than 107PFU/ml, more preferably 104～106PFU/ml, most preferably 105PFU/ml. The mode of infection of hACE2 mice after anesthesia preferably includes a nasal drip mode of infection. The infection dose of the virus liquid is preferably 45-55 mu L/virus liquid, and more preferably 50 mu L/virus liquid.
In the present invention, after infection, the mice are allowed to recover, preferably in sterile squirrel cages, to reduce contamination in the cages.
In the invention, 3-6 days after SARS-COV-2 virus strain infection, the mice are dissected. The time for observing the virus infection proliferation is preferably 3-4 d for infecting the virus strain. The time for observing lung lesions is preferably 5-6 days after the infection of the virus strain.
In the invention, a mouse model is constructed by adopting the construction method provided by the invention, and the mortality, weight change, clinical symptoms, virus titer, inflammatory factor expression condition and lung disease pathology examination condition of the mouse are respectively counted, and the results are as follows: when the virus strain is infected by 5 days, the death rate of the mouse reaches more than 95 percent, and the death rate of a mouse model can be obviously reduced after the Reidesvir is adopted for intervention, so that the Reidesvir drug intervention has a certain death protection effect; when the constructed mouse model is infected by the virus strain for 5d, the weight is obviously reduced, and the weight is obviously different from that of a normal group and a drug intervention group; when the constructed mouse model is infected by the virus strain for 5d, the phenomena of emphysema, shrugging, abdominal distension and the like occur; when the constructed mouse model is infected by the virus strain for 3d and 5d, the lung tissue homogenate virus titer is remarkably increased, and after drug intervention treatment, the lung virus titer is remarkably reduced; pulmonary hemorrhage, interstitial pneumonia, bronchial end degeneration/necrosis and desquamation of lung tissues of the mouse model. Therefore, the clinical symptoms of the mouse model constructed by the invention are consistent with the clinical symptoms of human infection with the new coronavirus, and simultaneously, the live virus titer detection reflects the real drug effect situation in the research of the vaccine and the antiviral drug, and plays a relatively important role in the preclinical research of the new coronavirus antiviral drug and the vaccine. Therefore, the invention provides the application of the mouse model of SARS-COV-2 virus infection constructed by the method in screening the medicine for resisting SARS-COV-2 virus or the vaccine for preventing and controlling SARS-COV-2 virus.
The construction method and application of SARS-COV-2 virus strain and mouse model provided by the present invention will be described in detail with reference to the following examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Method for separating SARS-COV-2 virus strain
1. Sample(s)
Pharyngeal swabs, nasopharyngeal swabs, sputum, bronchial/alveolar lavage, feces, urine, and the like. Samples were collected in Hank's medium (pH 7.4). The medium contained broad-spectrum antifungal and bacterial antibiotics (amphotericin B15 g/mL, penicillin 200IU/mL, streptomycin 200g/mL) and 1% BSA. And collecting the sample, subpackaging and freezing at-80 ℃.
2. Instrument, reagent and consumable
Vero E6 cells, cell culture plate, cell culture flask, 2mL screw-cap cryopreservation tube, DMEM medium, 2% bovine serum DMEM medium, broad-spectrum antifungal and antibacterial antibiotics (amphotericin B, penicillin, streptomycin), centrifuge with protective sleeve (or biosafety centrifuge), sealable protective box or self-sealing bag, optical inverted microscope, 37 ℃ 5% CO2Cell incubator, -80 ℃ refrigerator. Freshly prepared 2000mg/L chlorine-containing disinfectant solution.
3. Procedure for the preparation of the
(1) Vero E6 cells were subcultured in cell culture plates/bottles 18-24 h before.
(2) Taking a sample, melting the sample in a biological safety cabinet, adding a DMEM culture medium, stirring, and standing for 5-8 min.
(3) Transfer to a new sample tube and place in a protective sleeve for centrifugation at 2000rpm for 10 min. After centrifugation, the sample tube was opened in a biosafety cabinet, the supernatant was taken and transferred to a cell culture plate/bottle, and the remaining supernatant was separately frozen in a freezer at-80 ℃.
(4) The culture plate/bottle is put into a protective box and then is put into a cell culture box for culture for 3 hours. Shake gently every 15 min.
(5) And taking out the culture plate/bottle, adding sufficient 2% bovine serum DMEM medium into the biological safety cabinet according to the size of the culture container, and continuously culturing for 15 days. The solution was changed every 3 days. The culture supernatant can be stored in a refrigerator at-80 deg.C.
(6) Observation of SARS-CoV-2 cytopathic effect under microscope: cell shrinkage, rounding, fusion, death and shedding.
(7) If no significant cytopathic effect is observed, the culture supernatant can be harvested and serially passaged in Vero E6 cells for 2 to 3 passages in the same manner to observe the cytopathic effect of SARS-CoV-2.
The cells harvested and the culture supernatant were tested for SARS-CoV-2 virus strain in culture using RT-qPCR. The RT-qPCR detection reaction system is shown in the following table 1.
TABLE 1 RT-qPCR detection reaction System
The RT-qPCR reaction conditions are shown in Table 2 below.
TABLE 2 RT-qPCR reaction conditions
The genes detected by RT-qPCR, reference genes and amplification primers thereof are shown in Table 3.
TABLE 3 RT-qPCR primer sequence Listing
When the cells have obvious pathogenic effect or the virus nucleic acid detection is positive (CT value is less than 30), the culture container and the protective box are frozen and stored in a refrigerator at-80 ℃.
Example 2
Viral titer determination
1. Sample(s)
The novel coronavirus (SARS-CoV-2 culture sample) cell culture prepared in example 1 was frozen in a freezer at-80 ℃.
2. Instrument reagent consumptive material
Vero E6 cells, 96-well cell culture plate, 1.5mLEP tube, DMEM medium, antibiotics (penicillin and streptomycin), Gibco fetal bovine serum, sealable protective box, optical inverted microscope, 5% CO at 37 ℃2A cell culture box, a refrigerator at-80 ℃ and a freshly prepared 2000mg/L available chlorine solution.
3. Procedure for the preparation of the
(1) Subculturing the Vero E6 cells in a 96-well plate before 24 hours, and washing monolayer cells of the 96-well plate 1 time by using PBS;
(2) adding ten-fold diluted virus solution prepared in example 1, 100 mu L/hole, and incubating at 37 ℃ for 2 hours or centrifuging at 3000rpm for 1 hour;
(3) the supernatant was discarded, fresh virus culture was added at 100. mu.L/well, and the results were observed after 4 days, and CPE was recorded.
(4) Cytopathic effects were recorded and the results are shown in table 4.
TABLE 4 Virus-induced cytopathic Effect (CPE) grade 6 criteria
(5) Calculation of TCID by Karber method50Value, statistical treatment.
The virus titer TCID is measured50Value of 107PFU/mL。
Example 3
Method for constructing experimental model of SARS-COV-2 virus infected hACE2 mouse
1. Randomly selecting a K18hACE2 mouse with the age of 4-12 weeks and the weight of 23-35 g as an experimental animal, wherein the mouse can be used as both a male mouse and a female mouse.
2. All mice were infected in biosafety cabinets. The hACE2 transgenic mice are divided into a normal group (no infection), a SARS-CoV-2 infection group (model group), and a SARS-CoV-2 infection + Rudesevir drug intervention group, wherein each group comprises 8-10 mice. Anesthetizing with isoflurane (isoflurane), placing the plastic dryer in a biological safety cabinet, and adding isoflurane into the plastic dryer; the mice are placed in a dryer for anesthesia, and the respiration of the mice is observed until the anesthesia effect is achieved. Each set was set to 3 replicates.
3. Dilution of SARS-CoV-2 Virus: the cryopreserved tubes containing SARS-CoV-2 virus were removed from the-80 ℃ freezer and registered. Measuring the virus by using a pipettor into a freezing tube containing a proper amount of PBS or DMEM for dilution to obtain the titer of 107PFU/ml virus solution.
4. The anesthetized mice were removed and 50. mu.L of diluted virus was instilled into the nasal cavity, and the virus was instilled into the nasal drops of each group of mice except for the normal group given PBS. 2 hours after infection, the mice in the drug intervention group are administered with the Reidesciclovir by gavage, the administration amount of the Reidesciclovir is 50mg/kg, and the Reidesciclovir is continuously administered for 5 days and 1 time per day.
5. Mice were waited for recovery from anesthesia and placed in sterile squirrel cages to reduce possible intra-cage contamination.
Example 4
The mortality rate of the fifth day of infection of the SARS-CoV-2 infection group mouse model prepared in example 3 and the normal group mouse and the drug intervention group mouse were compared.
Mice in the model group, the normal group and the drug intervention group were counted for death at 5d infection.
As shown in the following Table 5, compared with the blank group, the mortality rate of the K18hACE2 mouse model infected with SARS-COV-2 new coronavirus is obviously increased (p is less than 0.001), and the mouse model has a certain death protection effect after drug intervention treatment.
TABLE 5 statistical results of mortality 5 days after infection in groups of mice
Note: denotes P <0.05 compared to the normal group, denotes P <0.01 compared to the normal group, denotes P <0.001 compared to the normal group.
Example 5
Continuously observing the weight change and clinical symptoms of a K18hACE2 mouse model infected with SARS-CoV-2 new coronavirus, normal mice and intervention mice for 5 days.
The results of the body weight changes of the mice in each group are shown in FIG. 1, and the body weight reduction trend of the K18hACE2 mouse model of SARS-CoV-2 new coronavirus is significantly different from the body weight reduction trend of each group.
The results of the dissection of clinical symptoms of the mice in each group are shown in FIG. 2. Clinical symptoms show that the K18hACE2 mouse model infected with SARS-CoV-2 new coronavirus has the phenomena of emphysema, shrugging hair, abdominal distension and the like, and the blank group of mice has no obvious clinical characteristics.
Example 6
Comparing the lung homogenate virus titer TCID on the third and fifth days of a K18hACE2 mouse model infected with SARS-CoV-2 new coronavirus, normal group mice and drug intervention group mice50The value is obtained. Virus titer TCID of homogenate50The values were calculated in the same manner as described in example 2.
Results as shown in table 6 below, the lung homogenate virus titer of SARS-CoV-2 neocoronavirus infected K18hACE2 mice was significantly increased (p <0.001) and significantly decreased (p <0.05) after drug intervention treatment compared to the blank group of mice.
TABLE 6 TCID for viable viral titre of lung homogenates 3 and 5 days after infection of groups of mice50Value of
Note: denotes P <0.05 compared to the normal group, denotes P <0.01 compared to the normal group, denotes P <0.001 compared to the normal group.
Example 7
Comparing the expression of lung tissue homogenate inflammatory factors (IL-1 beta, IFN-gamma, MCP-1 and IFN-alpha) of mice of an infected SARS-CoV-2 group mouse model, a normal group mouse and a drug intervention group mouse on the fifth day. The detection method of the inflammatory factors adopts a qPCR method for detection. The RT-qPCR detection reaction system is shown in Table 7 below.
TABLE 7 RT-qPCR detection reaction System
The RT-qPCR reaction conditions are shown in Table 8 below.
TABLE 8 RT-qPCR reaction conditions
The genes detected by RT-qPCR, reference genes and amplification primers thereof are shown in Table 9.
TABLE 9 RT-qPCR primer sequence Listing
As shown in FIG. 3, compared with the blank mice, the expression of IL-1 β, IFN- γ, MCP-1 and IFN- α inflammatory factors was significantly increased in the K18hACE2 mouse model group infected with SARS-CoV-2 new coronavirus (p <0.001), and the expression of each inflammatory factor was significantly decreased after drug intervention (p < 0.05).
Example 8
Comparing the lung pathology examination results of the mice of the SARS-CoV-2 group, the normal group and the drug intervention group on the fifth day. The lung tissue sections were H-E stained and microscopically observed under a 200-fold microscope and photographed.
The results are shown in FIG. 4. Wherein FIG. 4A shows moderate hemorrhage of lung, severe interstitial pneumonia, moderate bronchiolar mucosal epithelium degeneration/necrosis, desquamation of lung in mice of SARS-CoV-2 infected mouse model group; fig. 4B shows moderate hemorrhage of lung, moderate interstitial pneumonia, mild degeneration/necrosis, and desquamation of epithelium of bronchiole mucosa in drug intervention group mice. Compared with the blank group of mice, the lung tissues of the mice infected with SARS-CoV-2 mice model group have changes such as pulmonary hemorrhage, interstitial pneumonia, bronchial mucosa degeneration/necrosis, abscission and the like.
According to the detection results, the mortality rate of the hACE2 mouse model constructed by infecting the hACE2 mouse with the virus strain provided by the invention is up to 95.83% +/-7.22% on the 5 th day after infection, and the clinical symptoms of infection are close to the clinical reaction condition of human, which indicates that the mouse model is successfully constructed by the invention. Meanwhile, the titer of live viruses in the body of the mouse is detected in the research of the vaccine and the antiviral drug, so that the real drug effect condition can be reflected, and the method plays a relatively important role in the preclinical research of the new corona antiviral drug and the vaccine.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> Guangzhou medical university affiliated first hospital
GUANGZHOU INSTITUTE OF RESPIRATORY HEALTH
GUANGDONG LEWWIN PHARMACEUTICAL RESEARCH INSTITUTE Co.,Ltd.
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Claims (8)
1. A SARS-COV-2 virus strain, wherein the preservation number of the SARS-COV-2 virus strain is CCTCC NO: V202138.
2. Use of the SARS-COV-2 virus strain of claim 1 to construct a mouse model of SARS-COV-2 virus infection.
3. A method for constructing a mouse model infected by SARS-COV-2 virus is characterized by comprising the following steps:
after anesthetizing hACE2 mice, the anesthetized hACE2 mice were infected with a virus solution containing the SARS-COV-2 virus strain of claim 1.
4. The method of claim 3, wherein the titer of said strain of SARS-COV-2 virus in said viral fluid is not less than 107PFU/ml。
5. The method of constructing a mouse model of SARS-COV-2 virus infection as claimed in claim 4, wherein the titer of the strain of SARS-COV-2 virus is 104～106PFU/ml。
6. The method of constructing a mouse model of SARS-COV-2 virus infection as claimed in claim 3, wherein the mode of infection of the anesthetized hACE2 mouse comprises a nasal drip mode of infection.
7. The method for constructing a mouse model infected with SARS-COV-2 virus according to claim 3, wherein the infection dose of the virus solution is 45 to 55. mu.L/mouse.
8. The application of the mouse model of SARS-COV-2 virus infection constructed by the method of any one of claims 3 to 7 in screening drugs against SARS-COV-2 virus or vaccines for preventing and controlling SARS-COV-2 virus.
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