Construction method of mouse model infected by respiratory syncytial virus

Abstract

The invention belongs to the technical field of biology, and particularly relates to a construction method of a respiratory syncytial virus infected mouse model. Disodium clodronate liposome with the concentration of 4ng / mL is injected into the lateral ventricle of the mouse through stereotactic positioning of the brain. 48h after injection, mouse brain tissues are taken to detect the activation state of microglial cells through immunofluorescence and WB, after it is confirmed that the microglial cells are inhibited, RSV (virus titer: 2.8 * 10 < 6 > PFU) nasal drop infection experiments are carried out on the mouse, and after the 5th day after virus infection, the mouse is infected by the RSV (virus titer: 2.8 * 10 < 6 > PFU) nasal drop infection experiments. Likewise, mouse brain tissue is taken for virus load detection, and neuropathological changes of the mouse brain tissue are observed through HE staining, immunofluorescence and TUNEL staining, so that the method can be used for constructing a research model of RSV-related nervous system diseases.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a method for constructing a mouse model of respiratory syncytial virus infection. BACKGROUND

[0002] Human respiratory syncytial virus (hRSV) is an important pathogen causing acute lower respiratory tract infection (ALTRI) in newborns, children and the elderly. Severe hRSV infection can cause diseases such as bronchitis, pneumonia and respiratory failure. In recent years, studies have found that respiratory syncytial virus (RSV) also has significant neuroinvasive potential, can break through the blood-brain barrier and cause a series of pathological changes in the central nervous system (CNS), which is related to neurological sequelae. These manifestations are closely related to the proliferation and activation of brain tissue microglia cells found in autopsies, indicating that RSV infection not only affects the respiratory system, but also can have a profound impact on the central nervous system. Studies using mouse models have also confirmed that RSV can infect olfactory sensory neurons and achieve direct invasion of the central nervous system by entering the olfactory bulb; studies have also shown that RSV can infect neural cells through the lung-brain axis, leading to nerve damage. However, there is currently no effective vaccine, and its pathogenic mechanism remains to be studied.

[0003] Previous studies mostly used high-dose PLX 3397 (CSF1R inhibitor) or PLX 5622 (CSF1R inhibitor) in food to deplete 80-90% of microglia cells in the entire cortex. However, the systemic depletion method does not allow researchers to examine the role of microglia cells in specific brain regions, i.e., this method cannot be used to target specific brain tissues, can affect cell populations other than microglia cells, such as meningeal macrophages and microglia progenitor cells, and can have off-target effects on other cells such as microglia or oligodendrocyte progenitor cells; in addition, since CSF-1 receptor signaling is essential for neural progenitor cell differentiation and proliferation, this strategy is not ideal for use during critical periods of brain development. Liposomal clodronate sodium can selectively deplete microglia cells in vitro without affecting the activity, activation or proliferation of astrocytes or neurons, and is currently a hot research topic, but if clodronate sodium salt is free clodronate salt that is not encapsulated in liposomes, it can affect the number of astrocytes as well as microglia cells.

[0004] Therefore, a suitable animal model is essential for studying the brain pathological features and mechanisms related to RSV infection. SUMMARY

[0005] The present application provides a method for improving the replication level of RSV virus in the brain of mice and aggravating brain nerve damage. In recent years, the role of microglia in neuropathology has attracted more and more attention, especially in close relationship with viruses.

[0006] The present application provides a method for constructing a mouse model of respiratory syncytial virus infection, the steps of the method comprising: 1) injecting sodium clodronate liposomes into the lateral ventricle of the mouse brain by stereotactic positioning, and confirming whether the microglia of the mouse is inhibited after 48 hours; 2) performing a respiratory syncytial virus nasal infection experiment on the mouse whose microglia is inhibited in step 1); The titer of respiratory syncytial virus in the nasal infection experiment is 2.8x10 6 PFU, and the amount is 50 μL; 3) On the 5th day after the nasal infection experiment, a mouse model of respiratory syncytial virus infection is obtained after detection.

[0007] Further, the concentration of sodium clodronate liposomes is 4 ng / mL.

[0008] Further, step 1) is to confirm whether the microglia is inhibited by detecting the brain tissue of the mouse.

[0009] Further, the detection is virus load determination, pathological detection, immunofluorescence and apoptosis detection of the brain tissue of the mouse.

[0010] Advantages 1. The present application selectively targets the microglia in the CNS, and directly administers sodium clodronate liposomes into the brain, avoiding the influence of the peripheral macrophages by the way of systemic depletion of microglia (including long-term feeding with feed and intraperitoneal injection); at the same time, compared with the injection into the hippocampus and striatum, the ventricle has an advantage in drug diffusion.

[0011] 2. The way of systemic depletion of microglia takes a long time, especially the amount added to the feed is large, and these CSF1 inhibitors are expensive, which causes economic losses to a certain extent.

[0012] 3、RSV infected Balb / c mice can activate brain microglia cells. Inject 2-4 μL of 4 mg / ml (maximum solubility) disodium chloromethylene bisphosphonate liposomes (marked as day 0), and 48 hours later (day 2), the brain microglia cells are mostly cleared, indicating that the microglia cell inhibition efficiency is high. At this time, the RSV virus is infected, and 120 hours later (day 5), the replication level of RSV in the brain is significantly improved, and the brain neuropathy is aggravated. This method is applied in RSV infection for the first time in the world, and has the advantages of long-term, stability, high efficiency and repeatability. Only once injection of lateral ventricle can achieve the effect of clearing microglia cells, and effectively promote the infection of RSV, achieving a win-win effect. It has practicality and representativeness in studying the pathological characteristics and mechanism of RSV infection related to the brain.

[0013] 4、This method has great reference value for studying central nervous system diseases caused by other respiratory viruses (such as rhinovirus, metapneumovirus, influenza virus, coronavirus and adenovirus) other than RSV.

[0014] It should be understood that the above general description and the following detailed description are only exemplary and explanatory, and cannot limit the present application. BRIEF DESCRIPTION OF DRAWINGS

[0015] The above and other objects, features and advantages of the present application will become more apparent from the following detailed description when taken in conjunction with the accompanying drawings, in which like reference characters refer to like parts throughout the several views, and in which exemplary embodiments of the present application are shown.

[0016] Figure 1 WB (Western Blot, protein immunoblot) detects the expression of Iba1 protein of microglia cells. Compared with mice injected with PBS, the expression level of Iba1 protein of the group injected with disodium chloromethylene bisphosphonate liposomes is significantly reduced.

[0017] Figure 2 The immunofluorescence (green) detection result is shown in the figure A. After injecting disodium chloromethylene bisphosphonate liposomes into the brain ventricle, the number of Iba1 positive cells in the brain of this group of mice is significantly reduced. Figure B shows that the number of Iba1 positive cells in the brain of the mice injected with PBS is not different from that shown in figure C, which is the wild type (WT). A, B and C represent three groups respectively: disodium chloromethylene bisphosphonate liposome injection group, PBS injection group and wild type group.

[0018] Figure 3 The q-PCR detection result is shown in the figure. After the microglia cells are inhibited by disodium chloromethylene bisphosphonate liposomes, the viral load of RSV is significantly increased. The brain of the mice injected with PBS and disodium chloromethylene bisphosphonate liposomes respectively and then dropped with normal saline (control) does not detect the viral load.

[0019] Figure 4 The comparison between the microglia cell inhibition group and the control group (PBS) is shown in the following figure. The pathological symptoms are more obvious: a small amount of neurons in the brain tissue are visible, which are pyknotic, dark, have unclear nucleus-cytoplasm boundaries, and enhanced basophilic (black arrow); a large amount of microglial cell infiltration is visible (blue arrow); a medium area of necrotic focus, a large number of necrotic nerve cells, and nuclear fragmentation are visible (green arrow); and a small area of hemorrhage is occasionally visible (yellow arrow). The control group: a small amount of neurons in the brain tissue are visible, which are pyknotic, dark, have unclear nucleus-cytoplasm boundaries, and enhanced basophilic (black arrow); a small amount of microglial cell infiltration is visible (blue arrow).

[0020] Figure 5 A is the result of immunofluorescence staining: after the microglial cells are inhibited, the number of neurons (Neu) is reduced; B is the result of TUNEL staining: compared with the control group (PBS), the number of apoptotic cells is significantly increased. DETAILED DESCRIPTION

[0021] Embodiments of the present application will be described in more detail by referring to the attached drawings. Although embodiments of the present application are shown in the drawings, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided to make the present application more thorough and complete, and to fully convey the scope of the present application to those skilled in the art.

[0022] Mouse and cotton rat infection models are the most commonly used animal models for RSV vaccine and drug research and development. The susceptibility of cotton rats to RSV in the respiratory system is about 100 times that of Balb / c mice, and the replication of RSV in the lungs is much higher than that in Balb / c mice. However, the brain of the cotton rat is not infected by RSV, and the virus replication level in this part is very low, and it does not cause any pathological damage to the brain. On the contrary, although the virus replication level in the lungs of Balb / c mice is not as high as that of cotton rats, a low level of RSV replication is detected in the brain, and the microglial cells are activated to cause a certain degree of nervous system lesions, which may be due to the fact that RSV infection in cotton rats cannot pass through the blood-brain barrier, while RSV infection in Balb / c mice can pass through the blood-brain barrier to cause damage to the nervous system. Therefore, most of the research focusing on RSV-related nervous system diseases still mainly uses Balb / c mice, but the replication of RSV in Balb / c mice is semi-permitted, and the brain is also semi-permitted, and the virus replication level is not high, and the brain only has certain lesions. If the replication level of RSV in the brain and the nervous system lesions can be enhanced, the relationship between RSV infection and nervous system complications can be further explored, which is of great significance for perfecting the clinical spectrum of RSV infection, optimizing treatment strategies, and developing effective prevention measures.

[0023] Microglia, as a part of the innate immune system, builds the first line of defense against viral invasion in the central nervous system. In the disease model of viral infection, microglia plays a double-edged sword. On the one hand, as a kind of macrophage, microglia can be recruited around infected neurons to play a role in phagocytosis of pathogens, thereby limiting the spread and replication of viruses; on the other hand, after viral infection, microglia, as the main professional antigen-presenting cells (APCs) in the central nervous system, can recognize viral pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs), and can uptake and process viral antigens, activate CD8 + T cells via the MHC-I pathway to directly clear infected neurons, regulate the adaptive immunity of T cells in the central nervous system, and thus protect the host. In view of this, the application adopts to inhibit brain microglia to improve the replication level of RSV in the brain and aggravate neuropathology.

[0024] The application selects liposome-encapsulated clodronate, i.e. clodronate disodium liposomes, to allow clodronate to be selectively targeted to microglia for depletion, while preserving other cell types in the brain. So far, the depletion of microglia using clodronate disodium liposomes has only been carried out in a few studies, such as in neonatal rats, clodronate disodium liposomes were infused into the hippocampus, and microglia depletion began to appear after 5 days, and then began to reappear. In adult rats, infusion of clodronate disodium liposomes (5 μl) into the hippocampus can deplete microglia for at least 3-7 days after infusion. Intracerebroventricular infusion of 1 μL of clodronate disodium liposomes in neonatal rats (1 and 4 days after birth) reduced microglia by about 90% after about 1.5-2 weeks. As can be seen, different strains of mice use different ways to deplete microglia, and the results are also different. The animal model related to RSV infection of the nervous system currently basically uses Balb / c mice, and RSV reaches the peak of viral replication in Balb / c mice at 5 days after infection, which is a critical period for studying viral infection mechanisms and evaluating other drugs or vaccines. Therefore, according to the characteristics of viral infection of different ages, different species and different routes, the application adopts a short-term, high-efficiency and targeted brain tissue method to inhibit microglia in Balb / c mice, and infects RSV within a certain period of time, so as to achieve the effect of inhibited microglia and elevated viral replication level.

[0025] Example 1 Reagents and instruments: Hep-2 cells were purchased from Hangzhou Meisen Cell Biotechnology Co., Ltd.; ready-to-use anesthetic was purchased from Nanjing Aibei Biotechnology Co., Ltd.; clodronate disodium liposomes were purchased from Shanghai Shunna Biotechnology Co., Ltd.; stereotactic injection device was purchased from Shenzhen Ruiwode Life Technology Co., Ltd.; Hamilton microsyringe was purchased from Beijing Bolangning Technology Co., Ltd.; CO2 constant temperature incubator was purchased from Thermo Fisher Scientific, Inc.

[0026] The ready-to-use BCA protein concentration assay kit was purchased from Seven Innovation Biotechnology Co., Ltd.; universal tissue fixative and tissue autofluorescence quencher were purchased from Wuhan Saiweier Biotechnology Co., Ltd.; Co-IP / WB tissue / cell universal lysis buffer (containing protease inhibitor), UniTM SDS-PAGE rapid electrophoresis buffer (50X), ultrasensitive ECL chemiluminescence detection kit, UniTM SDS-PAGE rapid transfer buffer (50X), PAGE gel one-step preparation kit (10%), MinuteBlock rapid low background blocking buffer and pre-stained protein marker were purchased from Affinity Biotechnology Co., Ltd.; Beta Actin rabbit polyclonal antibody was purchased from Wuhan Sanying Biotechnology Co., Ltd.; HRP-labeled anti-rabbit IgG antibody was purchased from Cell Signaling Technology, USA; IBA-1 rabbit monoclonal antibody was purchased from Abcam, UK; DAPI staining reagent, EDTA antigen retrieval solution (pH 9.0), antifluorescence quenching mounting medium and environmentally friendly transparent dewaxing solution were purchased from Beijing Solarbio; clodronate disodium liposomes were purchased from Shanghai Shunna Biotechnology Co., Ltd.

[0027] 1. RSV virus amplification When Hep-2 cells reached approximately 85% confluence, they were inoculated with RSV virus at a multiplicity of infection (MOI) of 0.1. After incubation at 37°C for 2 hours, 14 mL of virus maintenance medium was added, and the cells were placed in an incubator for further culture. Cytopathic effect (CPE) was monitored daily. When CPE was observed in 80-90% of the cells in the culture flask, the virus was harvested. The culture flask was then subjected to two freeze-thaw cycles at -80°C. After these cycles, the cells were transferred to centrifuge tubes and centrifuged at 2000 rpm for 15 minutes. The supernatant was collected, and the clear virus suspension was aliquoted into 1.5 mL EP tubes (1 mL per tube) and stored at -80°C for subsequent experiments.

[0028] 2. RSV virus titer determination Dilute the Hep-2 cell suspension to 1×10⁻⁶. 5 Cells / mL were seeded at 100 μL per well in a 96-well plate, and then incubated in a CO2 incubator for 24 h. The viral titers were then serially diluted 10-fold using viral dilution buffer, each diluted to a final titer of 10. -1、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 、10 -8 、10 -9 、10 -10 , take out the 96-well plate, aspirate the culture medium, and wash three times with PBS. Add 100 μL of diluted virus solution to each well, and set the last two rows of the plate as normal control groups. Add 100 μL of pure culture medium to the control groups, and place them in a CO2 incubator for incubation. Observe the pathological changes regularly, and stop the experiment when the cells show obvious pathological changes. Record the infection of each empty well, and calculate the TCID50 value according to the ratio of pathological changes in wells of different dilutions. The TCID50 value is 4 x 10 6 (TCID50 / mL). After conversion, 4 x 10 6 (TCID50 / mL) is equal to 2.8 x 10 6 PFU / mL, that is, the virus titer is 2.8 x 10 6 PFU / mL, which is usually referred to as 2.8 x 10 6 PFU.

[0029] 3. Inhibition of microglial cells 5-6-week-old Balb / c mice are deeply anesthetized by intramuscular injection of 1.25% alverdine at a dose of 0.2 mL / 10 g, the skin is cut about 1 cm along the human suture on the head of the mouse to expose the skull, and then a brain stereotactic injection instrument is used for positioning. The needle is placed at the bregma point, and the left lateral ventricle is positioned at 0.8 mm backward along the midline and 0.5 mm leftward. Mark the drilling position, and then use a drilling device to drill vertically at the mark. The depth should not be too large (to prevent drilling into the brain tissue, stop when there is a sense of emptiness). A 5-μL volume of Hamilton microsyringe is used to suck 4 μL of clodronate liposomes, and the control group is injected with 4 μL of PBS by the same method. The needle is inserted to a depth of 2 mm, and then injected at a speed of 0.3 μL / min. After injection, the needle is left for 3 minutes before being withdrawn. The wound is sutured, and the mouse is placed in a cage for normal feeding after waking up.

[0030] 4. Western blotting 48 hours after injection of sodium clodronate liposome, the mice were deeply anesthetized with anesthetic, and the brain tissue of the mice was taken and placed in a 1.5 mL EP tube. The EP tube was placed on ice, and magnetic beads and RIPA lysis buffer (containing protease inhibitors) were added. The mixture was ground at 4°C until no lumps were formed. The mixture was centrifuged at 12000 rpm / min and 4°C for 10 min. The supernatant was taken for BCA protein concentration determination. The protein concentration was uniformly adjusted to 8 μg / μL. Then 5×SDS-PAGE protein loading buffer was added, and the mixture was boiled for 5 min. SDS-PAGE gel was prepared, and the sample was loaded. After running at 80V for 30 min, the voltage was adjusted to 120V and continued to run for 40-50 min. Then the membrane was transferred by semi-dry transfer instrument. After the membrane was blocked with fast blocking solution at room temperature for 20 min, the antibody was incubated at 4°C overnight. The membrane was washed with 1×TBST for 10 min each time, for a total of 3 times. The secondary antibody was incubated on a shaker at room temperature for 1 h. The membrane was washed with 1×TBST for 10 min each time, for a total of 3 times. The developing solution was applied to the NC membrane, and the image was taken. The results showed that compared with the mice injected with PBS, the expression level of microglial cell marker protein (Iba1) of the mice injected with sodium clodronate liposome was significantly reduced (p<0.01, Figure 5). Figure 1 ).

[0031] 5. Immunofluorescence 48 hours after injection of sodium clodronate liposome, the mice were deeply anesthetized with anesthetic, and the brain tissue of the mice was taken and placed in a 1.5 mL EP tube. The EP tube was placed on ice, and magnetic beads and RIPA lysis buffer (containing protease inhibitors) were added. The mixture was ground at 4°C until no lumps were formed. The mixture was centrifuged at 12000 rpm / min and 4°C for 10 min. The supernatant was taken for BCA protein concentration determination. The protein concentration was uniformly adjusted to 8 μg / μL. Then 5×SDS-PAGE protein loading buffer was added, and the mixture was boiled for 5 min. SDS-PAGE gel was prepared, and the sample was loaded. After running at 80V for 30 min, the voltage was adjusted to 120V and continued to run for 40-50 min. Then the membrane was transferred by semi-dry transfer instrument. After the membrane was blocked with fast blocking solution at room temperature for 20 min, the antibody was incubated at 4°C overnight. The membrane was washed with 1×TBST for 10 min each time, for a total of 3 times. The secondary antibody was incubated on a shaker at room temperature for 1 h. The membrane was washed with 1×TBST for 10 min each time, for a total of 3 times. The developing solution was applied to the NC membrane, and the image was taken. The results showed that compared with the mice injected with PBS, the expression level of microglial cell marker protein (Iba1) of the mice injected with sodium clodronate liposome was significantly reduced (p<0.01, Figure 5). Figure 2 ), indicating that the microglial cells were basically inhibited or removed by sodium clodronate liposome.

[0032] 6. RSV virus infection 48 hours after injection, 4×10 6 TCID50 / mL (titer of 2.8×10 6Balb / c mice were infected with 50ul of RSV virus (PFU) by intranasal instillation, and the mice injected with PBS and sodium chlorophosphate liposomes were instilled with the same volume of normal saline as control. After infection, the Balb / c mice were placed on their sides in the cage and allowed to eat freely. On day 5 after infection, the Balb / c mice were sacrificed, and the brain tissue was harvested for virus load determination, pathological examination, immunofluorescence, and TUNEL detection.

[0033] Example 2 Material sources: The RNA extraction kit (item number: RMA101-C2-P3) and the qPCR probe one-step kit were purchased from Nanjing Nuozhan Biotechnology Co., Ltd.; anhydrous ethanol, dimethylbenzene, and neutral gum were purchased from the National Pharmaceutical Group Chemical Reagent Co., Ltd.; HE staining reagent kit, DAPI staining reagent, EDTA antigen repair liquid (PH 9.0), anti-fluorescence quenching mounting agent, and environmentally friendly transparent deparaffinizing liquid were purchased from Beijing Solaybao; HRP-labeled anti-rabbit IgG antibody was purchased from Cell Signaling Technology, USA; neuron Neu rabbit monoclonal antibody was purchased from Abcam, USA.

[0034] 1. Virus load determination The RNA extracted from the brain tissue of the infected Balb / c mice constructed in Example 1 was subjected to fluorescent quantification by qPCR according to the one-step method. The 20ul qPCR reaction system was as follows: 2x One Step Q Probe Mix 10ul, One Step Q Probe Enzyme Mix 1ul, primer mixture 0.8ul, Probe 0.2ul, H2O 6.5ul, and template RNA 1.5ul.

[0035] The qPCR program was set as 50℃ reverse transcription for 15 min, 95℃ pre-denaturation for 30 s, and the cycle parameters were 95℃ for 10 s and 60℃ for 30 s. After 45 cycles, the fluorescence signal was collected for statistical analysis. The qPCR detection results showed that the brain tissue of the Balb / c mice injected with PBS could detect virus replication on day 5, with a virus copy number of 10 3.5 copies / mg, while the virus load of the mice injected with sodium chlorophosphate liposomes was 10 5.5 copies / mg, which was significantly higher than that of the mice injected with PBS (p<0.01). No virus load was detected in the brain of the control mice instilled with normal saline (Appendix Figure 3 ).

[0036] Table 1 qPCR primer sequences

[0037] 2. HE staining The mouse model constructed in Example 1 was deeply anesthetized with anesthetic, and then perfused with 4% paraformaldehyde. After the brain tissue was removed, the tissue was fixed with 4% paraformaldehyde for 48 h. The tissue was dehydrated, immersed in wax, and embedded after processing. The thickness of the section was 4 pm. Hematoxylin staining: the section was dyed in hematoxylin dyeing solution for 3-5 min, washed with tap water, differentiated with differentiation solution, washed with tap water, returned to blue with blue returning solution, and washed with running water. Eosin staining: the section was dehydrated in 95% alcohol for 1 min, dyed in eosin dyeing solution for 15 s, dehydrated and mounted, observed under a microscope, and the image was collected and analyzed.

[0038] From the HE staining of the brain tissue, it can be seen that the pathological symptoms after the challenge of the mouse injected with sodium chlorophosphate liposomes are more obvious than those of the mouse injected with PBS: a small amount of neurons can be seen in the brain tissue, which are pyknotic, deeply stained, have unclear nucleus-cytoplasm boundary, and enhanced basophilic property (black arrow); a large amount of microglial cell infiltration can be seen (blue arrow); a moderate area of necrotic focus, a large number of necrotic nerve cells, and nuclear fragmentation (green arrow) can be seen; and a small area of hemorrhage (yellow arrow) can be occasionally seen. The mouse group injected with PBS and then challenged: a small amount of neurons can be seen in the brain tissue, which are pyknotic, deeply stained, have unclear nucleus-cytoplasm boundary, and enhanced basophilic property (black arrow); a small amount of microglial cell infiltration can be seen (blue arrow) (see Figure 4 ).

[0039] 3. Immunofluorescence The immunofluorescence step of Example 1 is completely the same, except that the mouse (the mouse in this step is challenged with the virus) and the antibody (the primary and secondary antibodies in this step are both neuron (Neu) antibodies) are different. It can be known from the detection result that a large number of replicated viruses promote a significant decrease in the number of neurons (Neu) (p<0.01) after the inhibition of microglial cells (see Figure 5 , middle A).

[0040] 4. TUNEL staining The sectioning process is the same as the above HE staining step. The paraffin section is deparaffinized, soaked in PBS for 5 min, and repaired with proteinase K (after the section is slightly shaken dry, a circle is drawn around the tissue with a histological pen to prevent liquid flow). Incubate at 37°C for 20 min, then place the slide in PBS and shake on a shaker for 3 times, 5 min each time. After equilibration at room temperature for 10 min, add the reaction solution: mix an appropriate amount of TDT enzyme, dUTP, and buffer in a ratio of 2:5:50 to cover the tissue, and incubate in a 37°C incubator for 1 hour. DAPI restains the cell nucleus, and incubates at room temperature for 10 min in the dark. After sealing with an anti-fluorescence quenching mounting medium, the image is collected. The results show that the number of cell apoptosis is significantly up-regulated (p<0.01) (see Figure 5 , middle B).

[0041] The above results all show that after the microglia cells are inhibited, the virus replication level of RSV in the brain of Balb / c mice is obviously improved, and the damage to the central nervous system is also aggravated.

[0042] The application provides a high utilization value for studying the mechanism of central nervous system diseases caused by respiratory viruses other than RSV (such as rhinovirus, metapneumovirus, influenza virus, coronavirus and adenovirus) and developing new drugs.

[0043] The above has described the embodiments of the present application, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes are obvious to those skilled in the art without departing from the scope and spirit of the described embodiments. The selection of the terms used herein is intended to best explain the principles of the embodiments, practical application or improvement of the technology in the market, or to enable other ordinary skilled persons in the art to understand the embodiments disclosed herein.

Claims

1. A method for constructing a mouse model of respiratory syncytial virus infection, characterized by, The steps of the construction method include: 1) injecting sodium clodronate liposomes into the lateral ventricle of the mouse brain through brain stereotaxis, and confirming whether the microglia of the mouse is inhibited after 48 hours; 2) performing a respiratory syncytial virus nose drop infection experiment on the mouse whose microglia is inhibited in step 1); The titer of respiratory syncytial virus in the nasal drop infection experiment was 2.8 x 10 6 PFU, in an amount of 50 μL; 3) confirming the respiratory syncytial virus infected mouse model after 5 days after the nose drop infection experiment.

2. The construction method of claim 1, wherein, The concentration of the sodium clodronate liposome is 4 ng / mL.

3. The construction method of claim 1, wherein, The step 1) is to confirm whether the microglia is inhibited by detecting the brain tissue of the mouse.

4. The construction method according to claim 3, characterized in that, The detection is to detect the viral load, pathology, immunofluorescence and apoptosis of the brain tissue of the mouse.

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