Model construction method for Zika virus infected rats
By constructing the ZIKV challenge model of Ifnar1-deficient rats, the problem of insufficient rat models in the prior art was solved, and efficient ZIKV infection and inflammatory response were achieved, which was suitable for the ZIKV infection mechanism and drug development.
Patent Information
- Application Number
- CN202510563501.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-30
- Publication Date
- 2025-08-15
AI Technical Summary
The lack of effective Zika virus (ZIKV) rat model in the prior art limits the progress of ZIKV infection mechanism research and antiviral drug development. The mouse model is complex in operation and is difficult to meet the needs of drug toxicology and efficacy evaluation.
By inoculating rats with deletions of interferon alpha and β receptor subunit 1 (Ifnar1) to ZIKV, a ZIKV challenge animal model was constructed. An abdominal or intravenous injection was used to mainly detect blood, liver, spleen, reproductive organs and brain tissue.
The ZIKV infection model was efficiently constructed in rats, significantly increasing viral load and inflammatory response, spleen enlargement, spleen soma reduction, liver inflammatory cell infiltration, and upregulation of inflammation-related indicators. It is suitable for research on ZIKV infection mechanism and drug research and development.
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Figure CN120478423A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biotechnology, and in particular to a method for constructing a Zika virus-infected rat model. Background Art
[0002] Zika virus (ZIKV) is a mosquito-borne virus belonging to the genus Flavivirus in the family Flaviviridae. ZIKV particles are spherical, 40-70 nm in diameter, enveloped, single-stranded, positive-sense RNA viruses. There are two genotypes, Asian and African, and they are primarily transmitted through the bites of infected Aedes aegypti mosquitoes. ZIKV was discovered and documented over 70 years ago. Since the first major ZIKV outbreak in the Pacific Northwest in 2007, Zika has ravaged the globe, affecting approximately 80 countries and regions, with an estimated annual infection rate exceeding 2 million. In February 2016, the ZIKV outbreak was declared a Public Health Emergency of International Concern. my country also reported its first imported case of ZIKV in 2016, with sporadic cases subsequently occurring. While local transmission has not yet occurred in my country, the presence of ZIKV vectors such as Aedes albopictus and Aedes aegypti in my country suggests the possibility of local transmission remains, necessitating continued vigilance for ZIKV importations. ZIKV infection typically causes mild, flu-like symptoms, commonly including fever and rash. Some patients also experience conjunctivitis, muscle and joint pain, and general fatigue. Due to its high neurotropic nature, ZIKV infection during pregnancy may result in microcephaly in newborns, while infection in adults may cause Guillain-Barré syndrome. Currently, there is no safe and effective vaccine or specific anti-ZIKV drug to prevent or treat Zika virus infection.
[0003] Establishing an effective ZIKV challenge animal model is of great significance for studying the mechanism of ZIKV infection and developing specific anti-ZIKV drugs. Currently, mice lacking interferon receptors have been shown to be useful for establishing ZIKV challenge animal models, but rats have not yet been used in ZIKV-related research. As an important model animal, compared to mice, rats have behavioral characteristics similar to humans, a moderate body size, and are more convenient for drug administration, testing, and sample collection. They are easy to operate and observe, and are more suitable for drug toxicology, efficacy evaluation, new drug screening, and other studies. Therefore, it is of great significance to develop a new rat animal model that can be used for ZIKV challenge research. Summary of the Invention
[0004] In response to the above deficiencies in the prior art, the present invention provides a method for constructing a rat model of Zika virus infection, so as to establish an effective ZIKV challenge animal model for use in related research such as the infection mechanism of ZIKV and the development of specific anti-ZIKV drugs.
[0005] To achieve the above purpose, the specific technical solutions of the present invention are as follows:
[0006] In a first aspect, the present invention provides a method for constructing a rat model of Zika virus infection, wherein a ZIKV-challenged animal model is obtained by inoculating ZIKV into rats lacking interferon α and β receptor subunit 1 (Ifnar1).
[0007] Furthermore, the ZIKV inoculation dose is 1×10 4 PFU / mouse.
[0008] Furthermore, the vaccination method includes but is not limited to intraperitoneal injection or intravenous injection.
[0009] Furthermore, the intravenous injection includes but is not limited to tail vein injection.
[0010] Specifically, a ZIKV-challenged animal model was obtained by injecting ZIKV into the tail vein of rats lacking interferon α and β receptor subunit 1.
[0011] Furthermore, the samples tested in the ZIKV-challenged animal model are blood, liver, spleen, reproductive organs and brain tissue.
[0012] In a second aspect, the present invention provides a ZIKV challenged animal model obtained by the method.
[0013] In a third aspect, the present invention provides applications of the ZIKV challenge animal model in ZIKV infection mechanism research, vaccine development, or anti-ZIKV drug development.
[0014] Compared with the prior art, the present invention is beneficial in that:
[0015] The present invention establishes a ZIKV challenge animal model by inoculating rats lacking the interferon α and β receptor subunit 1 (Ifnar1). Compared to uninjected Ifnar1-deficient rats, ZIKV-inoculated rats exhibited significant ZIKV loads in the blood, liver, spleen, reproductive organs, and brain tissue. Furthermore, the spleen exhibited significant enlargement, decreased splenomegaly, and the liver showed extensive inflammatory cell infiltration. Inflammation-related markers, such as mRNA levels of TNF, Il6, Ccl5, Cxcl1, Isg15, and Isg56, were significantly upregulated. Furthermore, compared to mouse models, the challenge model presented in this invention had a high success rate and a shorter time to symptom onset. These results demonstrate that the ZIKV-infected Ifnar1-deficient rat challenge model presented in this invention is a novel, highly effective infectious animal model for low-pathogenicity viruses, useful for research into ZIKV infection mechanisms, vaccine development, and anti-ZIKV drug development. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] Figure 1 These are the ZIKV RT-PCR test results in the liver, spleen, reproductive organs, and brain tissues of young Ifnar1-deficient rats inoculated with ZIKV in Example 1;
[0017] Figure 2 This is a gross photograph of the spleen tissue of the young Ifnar1-deficient rats after inoculation with ZIKV in Example 1;
[0018] Figure 3 This is the HE staining result of the liver tissue of the young Ifnar1-deficient rats on the 16th day after inoculation with ZIKV in Example 1;
[0019] Figure 4 This is the HE staining result of spleen tissue of young Ifnar1-deficient rats on day 16 after inoculation with ZIKV in Example 1;
[0020] Figure 5 These are the results of detecting Tnf mRNA levels in the liver, spleen, reproductive organs, and brain tissues of young Ifnar1-deficient rats after inoculation with ZIKV in Example 1;
[0021] Figure 6 These are the results of detecting Ccl5 mRNA levels in the liver, spleen, reproductive organs, and brain tissues of young Ifnar1-deficient rats inoculated with ZIKV in Example 1;
[0022] Figure 7 These are the results of detecting Cxcl1 mRNA levels in the liver, spleen, reproductive organs, and brain tissues of young Ifnar1-deficient rats inoculated with ZIKV in Example 1;
[0023] Figure 8 These are the ZIKV RT-PCR test results in the blood, liver, spleen, reproductive organs, and brain tissues of adult Ifnar1-deficient rats inoculated with ZIKV in Example 2;
[0024] Figure 9 This is a gross photograph of the spleen tissue of adult Ifnar1-deficient rats after inoculation with ZIKV in Example 2;
[0025] Figure 10 This is the HE staining result of spleen tissue of adult Ifnar1-deficient rats on day 16 after inoculation with ZIKV in Example 2;
[0026] Figure 11These are the results of Il6 mRNA level detection in the liver, spleen, reproductive organs, and brain tissues of adult Ifnar1-deficient rats inoculated with ZIKV in Example 2;
[0027] Figure 12 These are the results of detecting Ccl5 mRNA levels in the liver, spleen, reproductive organs, and brain tissues of adult Ifnar1-deficient rats inoculated with ZIKV in Example 2;
[0028] Figure 13 The results of Isg15 mRNA level detection in the liver, spleen, reproductive organs, and brain tissues of adult Ifnar1-deficient rats inoculated with ZIKV in Example 2 are as follows;
[0029] Figure 14 These are the results of Isg56 mRNA level detection in the liver, spleen, reproductive organs, and brain tissues of adult Ifnar1-deficient rats inoculated with ZIKV in Example 2. DETAILED DESCRIPTION
[0030] The technical solutions of the present invention are described clearly and completely below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments obtained by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.
[0031] The present invention provides a method for constructing a rat model infected with Zika virus. The method comprises inoculating ZIKV into rats lacking interferon α and β receptor subunit 1 (Ifnar1) to obtain a ZIKV-challenged animal model.
[0032] In some examples, the vaccination method includes but is not limited to intraperitoneal injection or intravenous injection.
[0033] In some examples, the samples examined in the ZIKV-challenged animal model are blood, liver, spleen, reproductive organs, and brain tissue.
[0034] In the following specific examples, if specific experimental steps or conditions are not specified, the conventional experimental steps or conditions described in the literature in this field can be followed. The reagents or instruments used, if the manufacturer is not specified, are all conventional reagents and products that can be purchased on the market.
[0035] In the following specific examples, the ZIKV virus solution preparation method is as follows:
[0036] (1) The ZIKV strain was obtained from the School of Life Sciences, Wuhan University, and the strain number is ZIKA strain SZ-wiv01. After natural thawing, the strain was inoculated into a T75 culture flask containing C636 cells (cultured in DMEM medium (Gibco) containing 2% fetal bovine serum (Meilune)) at a growth density of approximately 80% and cultured in a 27°C, 5% CO2 incubator (Memmert, INCO153) for 3 days.
[0037] (2) After microscopic examination shows that half of the cells are floating, harvest. First, collect the supernatant in the culture flask into a 50 mL centrifuge tube and centrifuge at 3000 rpm and 4°C for 10 minutes. Leave about 2 mL of liquid in each culture flask. Use a cell scraper to scrape the cells in the culture flask and place them in another 15 mL centrifuge tube.
[0038] (3) Aspirate the supernatant after centrifugation with a syringe, then filter it through a 0.22 μm filter into a new centrifuge tube to obtain the virus solution, which is placed at 4°C for later use; use 15 mL of cell fluid to blow off the cell pellet after centrifugation, aspirate it into a 15 mL centrifuge tube, freeze it at -80°C or in liquid nitrogen, and then place it in a 37°C water bath to heat and dissolve it. Repeat this process 3 times.
[0039] (4) Place the thawed cell solution in a centrifuge and centrifuge at 3000 rpm and 4°C for 10 min. Aspirate the supernatant with a syringe and add it to the previously filtered virus solution. After mixing evenly, take 3 mL of the solution and divide it into 3 tubes for plaque assay. Then transfer it to a -80°C refrigerator for storage.
[0040] The ZIKV virus titer determination method is as follows:
[0041] (1) Vero E6 cells were cultured in a 37°C, 5% CO2 incubator to a growth density of approximately 90%. The culture medium was 1% methylcellulose. The culture medium was then aspirated and discarded. The cells were rinsed twice with serum-free DMEM. 1 mL of trypsin was added and the cells were placed in a CO2 incubator until the cells detached. The digestion time was up to 5 min.
[0042] (2) After digestion is complete, rinse the adherent cells with 2 mL of DMEM medium (supplemented with 10% fetal bovine serum and 1% antibiotics), then repeatedly pipette and vortex until the cells are no longer clumping. Then, add 10 mL of culture medium, tighten the cap, and shake the flask to mix the cells evenly.
[0043] (3) Add the mixed cell suspension to a 24-well plate, adding 0.5 mL to each well. Place the plate in a CO2 incubator and let it stand overnight. Observe the cells under a microscope and proceed to the next step after the cells adhere to the wall and fill each well.
[0044] (4) Prepare 6 EP tubes, add 900 μL of DMEM to each EP tube, take 100 μL of the virus stock solution and add it to the first EP tube, then pipette and mix it, then pipette 100 μL and add it to the second EP tube, repeat the previous step until all 6 EP tubes are operated. -1 to 10 -6 Six concentrations of virus solution were used.
[0045] (5) Transfer the culture plate from the incubator to the safety cabinet, aspirate the culture medium from the culture plate, and then add 500 μL of virus solution, adding four wells for each concentration. After completion, return the culture plate to the incubator and allow it to adsorb for 1-2 hours.
[0046] (6) After adsorption, remove the supernatant and discard it. Rinse the cells 2-3 times with DMEM. Add 500 μL of 1% methylcellulose culture medium to each well. Return the cells to the incubator and culture for 7 days.
[0047] (7) After the culture is complete, aspirate the culture medium and discard it. Rinse the cells 2-3 times with DMEM and then add 0.5-1 mL of fixative solution for at least 30 minutes. After fixation, aspirate the fixative solution and discard it. Stain with crystal violet solution for 10 minutes. Then rinse the cells slowly with running water to remove any excess stain.
[0048] (8) Calculate the number of plaques in the four wells at the minimum magnification that can be clearly seen by visual inspection, and then take the average value of the four wells.
[0049] (9) Calculate the virus titer according to the following formula:
[0050] Calculation formula: PFU / mL = average number of plaques / (dilution factor × volume of virus added per well).
[0051] Example 1 ZIKV challenge model in young rats with Ifnar1 gene deletion and effect evaluation
[0052] Ifnar1 gene-deficient rats were obtained from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd., and homozygous genotyping was completed at the Wuhan University Animal Experimental Center. The animal experiment project and protocol were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the Wuhan University Animal Experimental Center. All experiments were conducted in an ABSL-2 laboratory. 12-day-old Ifnar1 gene-deficient rats were divided into the following groups (D4, D7, D12, and D16, respectively, days 4, 7, 12, and 16 after virus injection):
[0053]
[0054] Each young Ifnar1 gene-deficient rat was intraperitoneally injected with ZIKV (1×10 4 PFU / mouse), and the control group was injected with the same volume of normal saline.
[0055] At the scheduled sampling time, rats were euthanized by CO2 asphyxiation. After death was confirmed by asphyxiation, the eyeballs were immediately removed to collect peripheral blood. The second step was to remove the genitals, with bilateral testicles removed from males and bilateral ovaries removed from females. The third step was to remove the spleen, perform gross measurements, and take photos. The fourth step was to remove the liver entirely, and perform nucleic acid testing on the right lobule. The fifth step was to remove the brain, cut the skull open along both sides of the occipital bone, and remove the brain with ophthalmic scissors.
[0056] RT-PCR was used to detect viral load in the testes, ovaries, spleen, liver, and brain to determine viral infection status in tissues. Relative quantification was calculated using 1000×2^-ΔCt (Ct experimental group minus Ct internal reference). The primers used for ZIKV detection were: F: 5'-TTGGT CATGA TACTG CTGAT TGC -3'; R: 5'-CCCTC CACGA AGTCTCTATT GC -3'. The test results are as follows: Figure 1 As shown, ZIKV was detected in the liver, spleen, brain tissue and reproductive organs of rats after infection.
[0057] The spleen tissue of rats in each group was observed. Figure 2 As shown, on the 12th and 16th days after infection, the spleen of rats showed obvious enlargement.
[0058] On the 16th day after infection, the liver and spleen of the rats were taken for pathological analysis. The HE staining results of the rat liver tissue were as follows: Figure 3 As shown in Figure 2, a large number of inflammatory cells infiltrated the portal area of the liver tissue of rats in the ZIKV infection group; the HE staining results of the spleen tissue of rats were as shown in Figure 2. Figure 4 As shown in the figure, the number of splenic bodies in spleen tissue was significantly reduced, the structure was disordered, and the number of lymphocytes was significantly reduced.
[0059] Finally, RT-PCR was used to detect the mRNA levels of inflammation-related molecules in liver, spleen, reproductive organs, and brain tissues, and 2^-△△Ct was used for relative quantitative multiple calculation. Figure 5 (Tnf), Figure 6 (Ccl5), Figure 7 As shown in Figure 3 (Cxcl1), the inflammation-related molecules Tnf, Ccl5, and Cxcl1 were significantly upregulated in various organs compared with the control group, and the inflammatory response was obvious, indicating that ZIKV infection was successful.
[0060] Example 2 ZIKV challenge model and effect evaluation of adult Ifnar1 gene-deficient rats
[0061] Ifnar1 gene-deficient rats were obtained from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd., and homozygous genotyping was completed at the Wuhan University Animal Experimental Center. The animal experiment project and protocol were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the Wuhan University Animal Experimental Center. All experiments were conducted in an ABSL-2 laboratory. Adult Ifnar1 gene-deficient rats were divided into the following groups (D4, D7, D12, D16, and D18, respectively, on days 4, 7, 12, 16, and 18 after virus injection):
[0062]
[0063] Each adult Ifnar1 gene-deficient rat was injected with ZIKV (1×10 4 PFU / mouse), and the control group was injected with the same volume of normal saline.
[0064] At the scheduled sampling time, rats were euthanized by CO2 asphyxiation. After death was confirmed by asphyxiation, the eyeballs were immediately removed to collect peripheral blood. The second step was to remove the genitals, with bilateral testicles removed from males and bilateral ovaries removed from females. The third step was to remove the spleen, perform gross measurements, and take photos. The fourth step was to remove the liver entirely, and perform nucleic acid testing on the right lobule. The fifth step was to remove the brain, cut the skull open along both sides of the occipital bone, and remove the brain with ophthalmic scissors.
[0065] RT-PCR was used to detect viral loads in serum, reproductive tissue, spleen, liver, and brain to determine viral infection status. Relative quantification was calculated using 1000×2^-ΔCt (Ct experimental group minus Ct internal reference). The primers used for ZIKV detection were: F: 5'-TTGGT CATGA TACTG CTGAT TGC -3'; R: 5'-CCCTC CACGA AGTCTCTATT GC -3'. The test results are as follows: Figure 8As shown in the figure, the characteristics of viral infection are that the number of viral copies increases with the extension of infection time, indicating that ZIKV forms persistent infection and viral replication in rats.
[0066] The spleen tissue of rats in each group was observed. Figure 9 As shown, on day 16 after infection, the spleen of rats showed obvious enlargement.
[0067] On the 16th day after infection, the spleen, brain and other tissues of rats in each group were taken for pathological analysis. The HE staining results of rat spleen tissue were as follows: Figure 10 As shown, some splenic corpuscles in the spleen tissue of rats in the ZIKV infection group had disordered structures.
[0068] Finally, RT-PCR was used to detect the mRNA levels of inflammation-related molecules in liver, spleen, reproductive organs, and brain tissues, and 2^-△△Ct was used for relative quantitative multiple calculation. Figure 11 (Il6), Figure 12 (Ccl5), Figure 13 (Isg15), Figure 14 As shown in Figure 3 (Isg56), inflammation-related molecules Il6, Ccl5, Isg15, and Isg56 were significantly upregulated in various organs compared with the control group, and the inflammatory response was obvious, indicating that ZIKV infection was successful.
[0069] In summary, the present invention establishes a ZIKV challenge animal model by inoculating rats lacking interferon α and β receptor subunit 1 (Ifnar1). Compared to rats lacking ZIKV that were not injected with ZIKV, significant ZIKV loads were detected in the rats' blood, liver, spleen, reproductive organs, and brain tissue after ZIKV inoculation. Furthermore, the spleen showed significant enlargement, splenomegaly decreased, and the liver tissue showed a large infiltration of inflammatory cells. The mRNA levels of inflammation-related indicators were significantly increased, indicating that the ZIKV challenge model of the present invention was successfully constructed and can be used in research related to ZIKV infection mechanisms, vaccine development, and anti-ZIKV drug development.
[0070] The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details of the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.
Claims
1. A method for constructing a Zika virus-infected rat model, characterized in that: A Zika virus-challenged animal model was obtained by inoculating rats lacking interferon α and β receptor subunit 1 with Zika virus.
2. The method for constructing a Zika virus-infected rat model according to claim 1, wherein: The inoculation dose of Zika virus is 1×10 4 PFU / mouse.
3. The method for constructing a Zika virus-infected rat model according to claim 1, wherein: The inoculation method includes but is not limited to intraperitoneal injection or intravenous injection.
4. The method for constructing a Zika virus-infected rat model according to claim 3, wherein: The intravenous injection includes but is not limited to tail vein injection.
5. The method for constructing a Zika virus-infected rat model according to claim 1, wherein: A Zika virus-challenged animal model was obtained by injecting Zika virus into the tail vein of rats lacking interferon α and β receptor subunit 1.
6. The method for constructing a Zika virus-infected rat model according to claim 1, wherein: The samples tested in the Zika virus-challenged animal model are blood, liver, spleen, reproductive organs and brain tissue.
7. A Zika virus challenged animal model obtained by the method according to any one of claims 1 to 6.
8. Use of the Zika virus-challenged animal model of claim 7 in the study of Zika virus infection mechanisms, vaccine development, or anti-Zika virus drug development.