RSV infection immortalized tree shrew aortic vascular endothelial cell model

Abstract

The invention discloses an RSV infection immortalized tree shrew aorta vascular endothelial cell model, and particularly relates to a method for separating and culturing primary aorta vascular endothelial cells from tree shrew aorta, further establishing an immortalized tree shrew aorta vascular endothelial cell line, taking the cell line as an RSV virus culture medium, and constructing an RSV infection immortalized tree shrew aorta vascular endothelial cell model. Establishing an RSV infected immortalized tree shrew aortic vascular endothelial cell model, and preparing a high-infection-titer RSV virus; the invention provides key support for preparation and detection of RSV virus and construction of a tree shrew in-vivo infection model, provides a stable virus source for development of comparative medical research or vaccine evaluation, and has far-reaching significance for deep exploration of RSV pathogenic mechanism, drug and vaccine evaluation and the like.

Description

Technical Field

[0001] The invention relates to a respiratory syncytial virus (RSV)-infected tree shrew aorta vascular endothelial cell model and an establishment method thereof, and belongs to the field of biotechnology. Background Art

[0002] Respiratory Syncytial Virus (RSV), belonging to the Paramyxoviridae family, is an important respiratory pathogen that carries single-stranded negative-sense RNA and has an envelope. The virus is particularly rampant in infants and the elderly, and can induce severe respiratory infection symptoms such as fever, cough, respiratory distress, and in extreme cases, even heart failure and death. Globally, tens of millions of children are attacked by RSV every year, and many of them unfortunately die. Unfortunately, despite the grim situation, there are currently only three RSV vaccines launched by GSK, Pfizer and Moderna on the market internationally, and my country is still blank in this field, with neither effective vaccines nor special drugs on the market, which greatly restricts the prevention and treatment of RSV infection.

[0003] Animal models play an indispensable role in the exploration of RSV infection disease characteristics and the development of drugs and vaccines. Although cotton rats, as a previously recognized RSV animal model, have been widely used in the analysis of disease mechanisms and the evaluation of drugs and vaccines, they have limitations in simulating human immune responses and disease manifestations. In addition, the current scarcity of cotton rat resources has seriously hindered the progress of RSV basic research and vaccine development. Therefore, it is particularly urgent to explore new animal models.

[0004] In addition, the in vitro culture system of RSV virus is also crucial, which is directly related to the successful construction of RSV infection model and the verification of vaccine efficacy. At present, RSV virus culture mainly relies on human laryngeal epidermoid carcinoma cells (Hep-2), monkey kidney cells (Vero), etc. However, these cells have problems such as long culture cycle, cumbersome titer determination and low titer, which makes it difficult to achieve significant improvement under the existing technical framework. Therefore, screening more suitable matrix cells for virus culture has become an important innovative strategy.

[0005] Tree shrew ( Tupaia belangeri ), as a close relative of primates, is highly similar to humans in terms of physiological and biochemical characteristics and genome structure. It is small in size, reproduces rapidly, has low breeding costs, and can be infected with a variety of human viruses, thus showing unique advantages in the study of viral infectious diseases. In recent years, tree shrews have shined in model studies of various viral infections such as hepatitis B virus, hepatitis C virus, influenza virus, etc., and have achieved fruitful results.

[0006] To verify the potential of tree shrews in RSV research, we made full use of the rich tree shrew cell resources and carried out RSV virus culture experiments on various tree shrew cells. The results showed that immortalized tree shrew aortic endothelial cells were highly sensitive to RSV. The cells exhibited a more typical cell syncytial pathological effect, a faster pathological progression, and were suitable for the simpler TCID50 virus titer determination method. Summary of the invention

[0007] The present invention provides an RSV in vitro infection model based on immortalized tree shrew aortic vascular endothelial cells, which solves the problems of long culture cycle, complex titer determination method and low titer of the existing RSV virus culture system, provides an experimental platform for the study of RSV infection mechanism, and also provides suitable viruses and technical support for the establishment of an RSV infection tree shrew in vivo model; using this model, RSV virus with high infection titer can be prepared, and at the same time, a specific virus load detection method and a convenient TCID50 virus titer determination method are established, which provide strong support for the accurate evaluation of RSV virus infection ability.

[0008] The present invention achieves the above object through the following technical solutions: 1. Isolation and culture of primary aortic endothelial cells from tree shrews Healthy tree shrews around 1 year old were selected and killed by intraperitoneal injection of 3% sodium pentobarbital for overdose anesthesia. The chest cavity was quickly cut open to expose the heart, and the heart was perfused using saline containing 1% penicillin-streptomycin. After the perfusion, the ascending aorta was straightened upward from the right atrial appendage, and the aortic arch segment was cut. The aortic arch was transferred to 4°C precooled saline containing 1% penicillin-streptomycin, rinsed repeatedly, the branches were cut off, and the residual connective tissue outside the blood vessels was removed. The aortic arch was cut longitudinally, the intima was turned outward, and cut into 0.8-1.2 mm small pieces. The tissue pieces were transferred to a T25 cell culture flask with forceps, and the intima side was attached to the wall. After standing for 2-5 minutes, the culture flask was inverted and 0.8-1 mL of 10% After culturing for 20 minutes at 37°C and 5% CO2, the culture flask was gently turned upside down to allow the tissue block to be immersed in the culture solution but not to float. After culturing for 12 hours, 2-3 mL of culture medium (DMEM / F-12 culture medium containing 10% FBS and 1% double antibody) was added. The culture was continued for several days, and fresh culture medium was replaced every 2 days. Cell growth was observed regularly. When the cell adhesion area around the tissue block reached 60-80%, 0.25% trypsin (containing EDTA) was used for the first digestion and subculture, followed by continuous subculture for 3-5 times. Cell morphology was observed under an inverted fluorescence microscope, and the characteristics of vascular endothelial cells were determined by immunofluorescence experiments of CD31 and vWF. 2. Establishment of immortalized tree shrew aorta endothelial cell line Primary tree shrew aortic endothelial cells in good growth condition were cultured at 2×10 5 The cells were inoculated in a 6-well plate at a density of 10 cells / mL, 2 mL per well, and cultured in a 37°C, 5% CO2 incubator. When the cell confluence reached more than 80%, the plates were washed twice with PBS, 1 mL of culture medium (DMEM / F12+1% double antibody+2% FBS) containing 4 μg / mL transfer agent (polybrene) was added, and the lentivirus carrying the SV40T gene was inoculated at an infection multiplicity (MOI) of 30. A control well of cells not inoculated with lentivirus was set up, and the plates were cultured in a 37°C, 5% CO2 incubator for 4 hours. After that, 1 mL of culture medium (DMEM / F12+1% double antibody+2% FBS) containing 4 μg / mL transfer agent was added and cultured continuously. After 24 hours, the plates were replaced with complete culture medium (DMEM / F12+1% double antibody+10% After 24 h, the culture medium was replaced with complete culture medium (DMEM / F12+1% double antibody+10% FBS) containing 4 μg / mL puromycin for continuous culture, and the complete culture medium containing 4 μg / mL puromycin was replaced once every 2 days. When the cells in the control wells died completely, the complete culture medium was replaced, and the surviving tree shrew aortic vascular endothelial cells transfected with SV40T were continuously subcultured for more than 50 generations, and the cell morphology and growth activity remained consistent, thus obtaining immortalized tree shrew aortic vascular endothelial cells.

[0009] 3. RSV infection of immortalized aortic endothelial cells in tree shrews Immortalized primary tree shrew aorta endothelial cells in good growth state were cultured at 2×10 6 The cells were inoculated into T25 cell culture flasks at a density of 10 cells / mL and cultured in a 37°C, 5% CO2 incubator until the cells were more than 80% confluent; in a biosafety cabinet, the cells were washed twice with PBS, and RSV virus of known titer from Hep-2 cells was inoculated at MOI=0.1. After incubation for 40-60 minutes, the virus solution was discarded and inactivated in 1% 84 disinfectant, and the cell culture flask was supplemented with 4 mL of culture medium (DMEM / F12+1% double antibody+2% FBS); the culture was continued in a 37°C, 5% CO2 incubator for several days, during which the cell pathological changes were observed every day, and the culture was terminated when about 80% of the cells were completely pathological; the mouth of the cell culture flask was sealed, placed in a -20°C refrigerator for more than 12 hours, thawed at room temperature, and repeated freezing and thawing twice; the virus culture was collected in a biosafety cabinet, centrifuged at 1000 rpm for 8 minutes, the cell fragments were deposited at the bottom, and the supernatant was taken to obtain the virus harvest solution.

[0010] 4. Immunofluorescence observation of RSV-infected immortalized aortic endothelial cells in tree shrews Immortalized aortic endothelial cells from tree shrews with good growth status were selected and cultured according to 10 5 cells / well were inoculated into a 24-well culture plate, and when the cells were about 80% full, the virus was inoculated and cultured in a biosafety cabinet according to step (3). After 48 h of virus culture, the culture medium was aspirated, the cells were rinsed once with PBS, and fixed with 4% paraformaldehyde for 20 min, and rinsed with PBS three times, each for 2 min. 0.5% Triton X-100 was added to perforate the cells for 20 min, and washed with PBS three times, each for 2 min. 3% BSA was used to block the cells for 30 min, and rinsed with PBS three times, each for 2 min. The diluted Anti-Respiratory Syncytial Virus antibody (ab43812) (1:500 dilution) was added, and the cells were incubated at 4°C overnight, and rinsed with PBS three times, each for 2 min. The secondary antibody was incubated at 37°C for 60 min, and rinsed with PBS three times, each for 2 min. The diluted DAPI staining solution was added to the culture wells, and the cells were incubated at room temperature in the dark for 2 min, and washed with PBS three times, 2 min each time; observed and photographed under a fluorescence microscope, and typical cell syncytial lesions were visible in the field of view.

[0011] 5. Virus proliferation curve of RSV-infected immortalized aortic endothelial cells in tree shrews Primers and probes were designed based on RSV conservative sequences as follows: F: 5'-AGGTGGTGAAGCAGGATTCTAC-3'; R: 5'-TGATTCCTCGGTGTACCTCTG-3'; P: 5'-FAM-CTCTCCCATTATGCCTAGGCCAGCGGC-BHQ1-3'; Immortalized aortic endothelial cells from tree shrews with good growth status were selected and cultured at a rate of 2×10 5cells / well were inoculated into a 12-well culture plate, and when the cells were about 80% full, the virus was inoculated and cultured in a biosafety cabinet according to the method in step (3); samples were collected in a biosafety cabinet at 12 h, 24 h, 48 h, 72 h, 96 h, and 120 h after infection, and RNA was extracted according to the instructions of the Takara RNAiso Easy Kit (TCH021); viral load was detected according to the Takara One Step PrimeScript RT-PCR Kit (DRR064A), and the reaction conditions were reverse transcription at 42 °C for 30 min, denaturation at 95 °C for 2 min, and amplification at 95 °C for 5 s and 58 °C for 30 s for 40 cycles; viral proliferation curves were drawn based on the viral load at each time point.

[0012] 6. Determination of virus titer in immortalized aortic endothelial cells infected with RSV Immortalized aortic endothelial cells from tree shrews with good growth status were selected and cultured at a rate of 5×10 4 cells / well were inoculated in a 96-well culture plate and cultured until the cells basically covered the bottom of the well; in a biosafety cabinet, the RSV virus harvest solution was diluted 10-fold in a gradient series, i.e., 10 -1 100 μL of virus harvest solution was added to 900 μL of culture medium (DMEM / F12+1% double antibody+2% FBS), and then the virus dilution solution was added to each gradient at 100 μL / well, 8 wells were paralleled, and a cell control group without virus infection was set up; the culture was continued for 7 days, during which the cytopathic condition was regularly observed and recorded, and the virus titer was calculated according to the Reed-Muench method.

[0013] Compared with Hep-2 cells and other immortalized tree shrew cells, the RSV-infected tree shrew immortalized aortic endothelial cell model established by this method has a faster and more typical cell syncytial pathological effect after virus infection, which can reduce the virus culture time and save time and materials. At the same time, after the virus is harvested, the established specific qPCR method can be used to quickly evaluate the virus proliferation. The TCID50 virus titer determination method can be used to quickly evaluate the virus infection activity, and compared with the plaque assay method, the operation is simpler, the technical difficulty is lower, and the repeatability is good. It provides key support for the preparation of RSV virus and the construction of the in vivo infection model of tree shrews, and provides a stable virus source for comparative medical research or vaccine evaluation. It has far-reaching significance for the in-depth exploration of RSV pathogenic mechanism, drug and vaccine evaluation, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1The morphology of primary culture and subculture of tree shrew aorta vascular endothelial cells. In the figure, P0 is the morphology of primary culture of tissue block adherence method for 9 days; P1 is the morphology of cells cultured for 2 days after subcultured once, and P4 is the morphology of cells cultured for 2 days after subcultured for 4 times; Figure 2 This is the immunofluorescence observation result of primary aortic endothelial cells of tree shrews; Figure 3 The morphology of tree shrew aorta vascular endothelial cells of different generations. In the figure, P10 is the morphology of the 10th generation of cells; P32 is the morphology of cells after the 32nd generation, and P60 is the morphology of cells after the 60th generation; Figure 4 This is the growth curve of aortic endothelial cells of tree shrews of different generations; Figure 5 The figure shows the cytopathic effect of RSV infection on various cells. In the figure, dpi is days after infection. TSLFC is immortalized tree shrew lung fibroblasts; TSVSMC is immortalized tree shrew vascular smooth muscle cells; TSVEC is immortalized tree shrew aorta vascular endothelial cells; Hep-2 is human laryngeal epidermoid carcinoma cell; Vero is monkey kidney cell. Figure 6 This is the immunofluorescence observation of RSV-infected tree shrew aorta vascular endothelial cells; Figure 7 The amplification curve (A) and standard curve (B) of RSV viral RNA by qPCR; Figure 8 This is the proliferation curve of RSV virus on different cells, including TSLFC immortalized tree shrew lung fibroblasts; TSVSMC immortalized tree shrew vascular smooth muscle cells; TSVEC immortalized tree shrew aorta vascular endothelial cells; Hep-2 human laryngeal epidermoid carcinoma cells; Vero monkey kidney cells; Fig. 9 RSV virus 10 -6 Cytopathic effects of 8 parallel wells infected with gradient dilution (7 dpi). DETAILED DESCRIPTION

[0015] The present invention is further described in detail below by way of examples, but the protection scope of the present invention is not limited to the contents described above. The methods in the examples are conventional methods unless otherwise specified, and the reagents used are conventional commercially available reagents or reagents prepared according to conventional methods unless otherwise specified. The percentage contents in the examples are all percentage contents by mass volume unless otherwise specified; Example 1: Isolation and culture of primary aortic endothelial cells from tree shrews 1. Take healthy tree shrews around 1 year old, inject 0.5 mL of 3% sodium pentobarbital intraperitoneally to kill them by overdose anesthesia, quickly cut the chest cavity to expose the heart, and perform cardiac perfusion with saline containing 1% penicillin-streptomycin; after the perfusion, straighten the ascending aorta from the right atrial appendage upwards, cut the aortic arch segment; transfer to 4℃ pre-cooled saline containing 1% penicillin-streptomycin, rinse repeatedly, cut off the branches, and remove the residual connective tissue outside the blood vessels; cut the aortic arch longitudinally, turn the intima outside, and cut it into 0.8-1.2 mm small pieces; transfer the tissue block to a T25 cell culture flask with tweezers, let the intima side adhere to the wall, let it stand for 5 minutes, then invert the culture flask, add 1 mL of 10% After culturing for 20 minutes at 37°C and 5% CO2, the culture flask was gently turned upside down to allow the tissue block to be immersed in the culture solution but not to float. After culturing for 12 hours, 2 mL of culture medium (DMEM / F-12 culture medium containing 10% FBS and 1% double antibody) was added. The culture was continued for several days, and fresh culture medium was replaced every 2 days. Cell growth was observed regularly. When the cell adhesion area around the tissue block reached 80%, 0.25% trypsin (containing EDTA) was used for the first digestion and subculture, and then subcultured for 4 consecutive times. The cell morphology was as follows: Figure 1 shown.

[0016] 2. Immunofluorescence identification of primary aortic endothelial cells in tree shrews Select cells with good growth status, digest and subculture them in 12-well culture plates, and wait until about 80% of the cells cover the bottom of the culture wells; discard the culture medium, rinse with PBS once, add 4% paraformaldehyde to fix for 20 minutes, rinse the well plate with PBS three times, each time for 3 minutes; add 0.5% Triton X-100 to perforate the cells for 20 minutes, rinse with PBS three times, each time for 3 minutes; use 2% BSA to block for 30 minutes, rinse with PBS three times, each time for 3 minutes; add diluted primary antibody, place at 4°C for overnight incubation; rinse with PBS three times, each time for 3 minutes; operate in the dark, add the corresponding secondary antibody to the culture well, incubate at 37°C for 60 minutes, rinse with PBS once, each time for 3 minutes; add diluted DAPI staining solution to the culture well, incubate in the dark for 2 minutes, wash with PBS three times, each time for 3 minutes; observe and take pictures under a fluorescence microscope. The results of immunofluorescence identification are shown in Figure 2 The cells were generally positive for CD31 and vWF, indicating that the cells obtained in Example 1 of the present invention were tree shrew aortic endothelial cells.

[0017] Example 2: Establishment of immortalized tree shrew aorta vascular endothelial cell line 1. Primary tree shrew aortic endothelial cells in good growth condition were cultured at 2×10 5The cells were inoculated in a 6-well plate at a density of cells / mL, 2 mL per well, and cultured in a 37°C, 5% CO2 incubator. When the cell confluence reached more than 80%, the plates were washed twice with PBS, 1 mL of culture medium (DMEM / F12+1% double antibody+2% FBS) containing 4 μg / mL transfer agent (polybrene) was added, and the lentivirus carrying the SV40T gene was inoculated at an infection multiplicity (MOI) of 30. A control well of cells not inoculated with lentivirus was set up and cultured in a 37°C, 5% CO2 incubator. After 4 hours, 1 mL of culture medium (DMEM / F12+1% double antibody+2% FBS) containing 4 μg / mL transfer agent was added for continued culture. After 24 hours, the culture medium was replaced with a complete culture medium without transfer agent (DMEM / F12+1% double antibody+10% FBS) for continued culture. After 24 hours, the culture medium was replaced with a complete culture medium containing 4 μg / mL puromycin for continued culture, and the culture medium was repeated every 2 hours. d. Replace the complete culture medium containing 4 μg / mL puromycin once; when the cells in the control wells are completely dead, replace the complete culture medium again, and culture the surviving tree shrew aortic endothelial cells transfected with SV40T for more than 50 generations, and observe that the cell morphology remains basically the same ( Figure 3 ), confirmed that immortalized tree shrew aortic endothelial cells were obtained.

[0018] 2. Detection of cell viability at different generations Prepare cell suspension and inoculate in 96-well culture plates at a concentration of 5 × 10 4 cells / mL, 100μL per well, set up experimental group and blank group at the same time, 3 replicates per group, set up 7 groups for 1-7 days; culture in a 37℃, 5% CO2 incubator, take out the culture plate at the same time point every day, remove the culture medium and wash twice with PBS, add 100μL of mixed solution containing CCK-8 reagent (90μL culture medium, 10μL CCK-8 reagent) to each well, and continue to culture for 30min; select wavelength 450nm, measure the absorbance (A value) of each well on the microplate reader, and draw the cell growth curve according to the absorbance value at each time point; the results are shown in Figure 4 As shown, each generation of cells entered the logarithmic growth phase at 3d-5d in culture, and the P60 generation cells still maintained a growth activity close to that of P10.

[0019] Example 3: Establishment of RSV-infected Tree Shrew Immortalized Aortic Vascular Endothelial Cell (TSVEC) Model Immortalized primary tree shrew aorta endothelial cells with good growth status were cultured in complete culture medium (DMEM / F12+1% double antibody+10% FBS) at a rate of 2×10 6The cells were inoculated into T25 cell culture flasks at a density of 10 cells / mL and cultured in a 37°C, 5% CO2 incubator until the cells were more than 80% confluent; in a biosafety cabinet, the cells were washed twice with PBS, and RSV virus of known titer from Hep2 cells was inoculated at MOI=0.1. After incubation for 50 minutes, the virus solution was discarded and inactivated in 1% 84 disinfectant, and the cell culture flask was supplemented with 4 mL of culture medium (DMEM / F12+1% double antibody+2% FBS); the culture was continued in a 37°C, 5% CO2 incubator for several days, during which the cell pathological changes were observed every day, and the culture was terminated when about 80% of the cells were completely pathological; the mouth of the cell culture flask was sealed, placed in a -20°C refrigerator for more than 12 hours, thawed at room temperature, and frozen and thawed twice; the virus culture was collected in a biosafety cabinet, centrifuged at 1000rpm for 8 minutes, the cell fragments were deposited at the bottom, and the supernatant was taken to obtain the virus harvest solution.

[0020] Immortalized aortic endothelial cells from tree shrews with good growth status were selected and cultured according to 10 5 cells / well were inoculated in a 24-well culture plate, and when the cells were basically full of about 80%: in a biosafety cabinet, virus inoculation and culture were performed according to the method described in the first paragraph of Example 5; after 48 hours of virus culture, the culture medium was aspirated and discarded, and the cells were rinsed once with PBS, fixed with 4% paraformaldehyde for 20 minutes, and rinsed with PBS three times, each time for 2 minutes; 0.5% Triton X-100 was added to perforate the cells for 20 minutes, and washed with PBS three times, each time for 2 minutes; 3% BSA was used for blocking for 30 minutes, and PBS was rinsed three times, each time for 2 minutes; diluted Anti-Respiratory Syncytial Virus antibody (ab43812) (1:500 dilution), incubate overnight at 4℃, rinse 3 times with PBS, 2 min each time; incubate with secondary antibody in dark conditions, incubate at 37℃ for 60 min, rinse 3 times with PBS, 2 min each time; add diluted DAPI staining solution to the culture wells, incubate for 2 min at room temperature in dark conditions, wash 3 times with PBS, 2 min each time; observe and take pictures under a fluorescence microscope.

[0021] At the same time, human laryngeal epidermoid carcinoma cells (Hep-2 cells), monkey kidney cells (Vero cells), immortalized tree shrew lung fibroblasts (TSLFC), and immortalized tree shrew vascular smooth muscle cells (TSVSMC) were infected with RSV as controls; The results are as follows Figure 5As shown, RSV can infect the aortic vascular endothelial cells, vascular smooth muscle cells and lung fibroblasts of tree shrews, but the syncytia of immortalized aortic vascular endothelial cells (TSVEC) cultured for 2 days is more obvious than that of the other two immortalized cells of tree shrews. A large number of cell deaths and floating cell clusters can be seen on the 5th day. Overall, the lesions of RSV-infected tree shrew aortic vascular endothelial cells are the most typical, similar to Hep-2 and Vero, and easier to identify. Immunofluorescence observation of RSV-infected tree shrew aorta endothelial cells was performed. Figure 6 As shown, RSV-infected cells are marked with green fluorescence, the cell nuclei are blue, and a large number of cell nuclei are aggregated in the syncytium area, which is consistent with the pathological changes observed previously, indicating that the present invention has successfully established an RSV-infected tree shrew immortalized aortic endothelial cell model.

[0022] Example 4: Determination of RSV viral load in immortalized tree shrew aorta endothelial cell culture Primers and probes were designed based on RSV conservative sequences as follows: F: 5'-AGGTGGTGAAGCAGGATTCTAC-3' R: 5'-TGATTCCTCGGTGTACCTCTG-3' P: 5'-FAM-CTCTCCCATTATGCCTAGGCCAGCGGC-BHQ1-3'; Immortalized aortic endothelial cells from tree shrews with good growth status were selected and cultured at a rate of 2×10 5 cells / well were inoculated in a 12-well culture plate until the cells were basically grown to about 80%: in a biosafety cabinet, virus inoculation and culture were performed according to the method of Example 3; samples were collected in a biosafety cabinet at 12h, 24h, 48h, 72h, 96h, and 120h after infection, and RNA was extracted according to the instructions of the Takara RNAiso Easy Kit (TCH021); viral load was detected according to the Takara One Step PrimeScript RT-PCR Kit (DRR064A), and the reaction conditions were reverse transcription at 42°C for 30min, denaturation at 95°C for 2min, and amplification at 95°C for 5s and 58°C for 30s for 40 cycles; viral proliferation curves were drawn according to the viral load at each time point; Hep-2 cells were set as a control in the experiment; the amplification results are shown in Figure 7 As shown, the amplification curve presents a standard "S" shape, and the gradients are well parallel; the standard curve E = 100%, R 2 =0.999, slope =3.322, indicating that the detection method is accurate and the test results are reliable; the virus proliferation curve is drawn according to the copy number of viral RNA at different time points.

[0023] In order to further compare the replication ability of RSV virus on different cells, under the same multiplicity of infection, this example plotted the proliferation curve of the virus on different cells with reference to the above method. The results are as follows: Figure 8 As shown in the figure, the proliferation trend of RSV in tree shrew immortalized aortic vascular endothelial cells (TSVEC) is basically the same as that in Hep-2, and the replication ability is stronger than other tree shrew immortalized cells, with the highest load reaching 7.8×10 7 copies / mL. In general, immortalized tree shrew aortic endothelial cells are very suitable for the culture of RSV virus.

[0024] Example 5: Determination of virus titer in immortalized aortic endothelial cells infected with RSV Immortalized aortic endothelial cells from tree shrews with good growth status were selected and 5×10 4 cells / well were inoculated in a 96-well culture plate and cultured until the cells basically covered the bottom of the well; in a biosafety cabinet, the RSV virus harvest solution was diluted 10-fold in a gradient series, i.e., 10 -1 100 μL of virus harvest solution was added to 900 μL of culture medium (DMEM / F12+1% double antibody+2% FBS), and then 100 μL / well of virus dilution was added to each gradient, 8 wells were paralleled, and a control group of cells not inoculated with virus was set up; culture was continued for 7 days, during which the cytopathic condition was regularly observed and recorded, and the virus titer was calculated according to the Reed-Muench method: First, the difference between the logarithm of the dilution above 50% infection rate (a) and the logarithm of the dilution below 50% infection rate (ba) was calculated; Then calculate the distance ratio x = (percentage above 50% infection rate - 50%) / (percentage above 50% infection rate - percentage below 50% infection rate); Finally, substitute a, x and the logarithmic difference of the dilution into the formula TCID50=10^(a+x×(ba)).

[0025] The cytopathic conditions in this example are shown in Table 1 and Fig. 9 As shown, a=6, b=7, x=0.5, TCID50=10 6.5 , the virus volume added to each well was 100 μL, and the final calculated virus titer = 10 7.5 TCID50 / mL, indicating that RSV can maintain strong infection activity on immortalized aortic endothelial cells of tree shrews, and the TCID50 detection method is suitable for the quick determination of virus titer.

[0026] Table 1 Statistics of cytopathic effect (CPE) caused by RSV infection 。

Claims

1. A model of RSV infection of immortalized tree shrew aortic endothelial cells, characterized in that: Primary aortic endothelial cells were isolated and cultured from the aorta of tree shrews, and an immortalized tree shrew aortic endothelial cell line was further established. Using this immortalized cell line as a virus culture matrix, a respiratory syncytial virus (RSV) infection experiment was conducted to successfully construct a model of RSV-infected immortalized tree shrew aortic endothelial cells, and the preparation of RSV virus with high infectious titer was achieved.

2. The RSV-infected immortalized tree shrew aortic vascular endothelial cell model according to claim 1, characterized in that: The immortalized tree shrew aortic endothelial cells in this model showed high sensitivity to respiratory syncytial virus. After infection with RSV, these cells showed typical cellular syncytial pathological effects, and the pathological process was more rapid.

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