Establishment method and application of Zika virus infectious animal model

Abstract

The invention provides an establishment method and application of a Zika virus infectious animal model, and belongs to the technical field of biology. According to the invention, ZIKV is inoculated to Toll-like receptor-deleted animals, such that the ZIKV challenging animal model is obtained. After the animal is inoculated with ZIKV, obvious ZIKV load can be detected in blood, liver, spleen, reproductive organ and brain tissue of the animal, moreover, the spleen is obviously swollen, the liver tissue has punctiform necrotic lesion, the splenosome is obviously reduced, and the mRNA level of inflammation related indexes Ccl5, Cxcl1, Isg15 and Isg56 is obviously up-regulated, which indicates that the ZIKV challenge model disclosed by the invention is successfully constructed; the method can be used in related researches such as ZIKV infection mechanism, vaccine research and development, anti-ZIKV drug research and development and the like.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for establishing a Zika virus infectious animal model and its application. 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, and are 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. Due to the presence of ZIKV vectors such as Aedes albopictus and Aedes aegypti in my country, local transmission of ZIKV remains a possibility, and ZIKV importation is a concern. 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 neurotropism, ZIKV infection during pregnancy may result in microcephaly in newborns, while infection in adults may cause Guillain-Barré syndrome.

[0003] ZIKV has a complex pathogenesis. After infecting a host, it first enters cells, particularly macrophages and neurons, by binding to host cell surface receptors. This activates the host immune system and generates an inflammatory response, but the detailed infection mechanisms remain to be determined. Currently, there are no approved clinically effective anti-ZIKV drugs or vaccines for ZIKV use. Therefore, establishing effective animal models of ZIKV infection is crucial for elucidating the viral pathogenesis and accelerating the development of vaccines and antiviral drugs. Similar to dengue virus, ZIKV does not cause infection or disease in wild-type adult mice. Dick et al. tested ZIKV infection in several animal species, including mice, cotton rats, guinea pigs, and rhesus macaques. Their results showed that only neonatal mice were susceptible to symptomatic ZIKV infection. Subsequent studies have demonstrated ZIKV infection and disease symptoms in rhesus macaque and chicken embryos. However, neither neonatal mice nor animal embryos are suitable for the development of ZIKV pathogenicity and preventive drugs. To overcome this limitation, it is urgent to develop methods to construct ZIKV-challenged animal models that can achieve effective ZIKV infection, so as to be used in related research on ZIKV infection mechanism, vaccine development, and antiviral drug development. Summary of the Invention

[0004] In response to the above deficiencies in the prior art, the present invention provides a method for establishing an animal model of Zika virus infection, so as to establish an effective ZIKV-infected animal model for use in research related to ZIKV infection mechanism, vaccine development, and antiviral drug development.

[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 establishing a Zika virus infectious animal model, wherein a ZIKV-challenged animal model is obtained by inoculating a Toll-like receptor (TLR)-deficient animal with ZIKV.

[0007] Furthermore, the TLR-deficient animal includes: TLR-deficient mice, TLR-deficient rats, TLR-deficient guinea pigs, TLR-deficient hamsters, TLR-deficient rabbits, TLR-deficient dogs or TLR-deficient monkeys.

[0008] Furthermore, the TLR-deficient animal is a TLR3-deficient animal or a TLR7-deficient animal.

[0009] Furthermore, the ZIKV vaccination dose is 1 × 10 4 PFU / each ~1 × 10 5 PFU / mouse.

[0010] Furthermore, the vaccination method includes but is not limited to intravenous injection.

[0011] Furthermore, the intravenous injection includes but is not limited to tail vein injection.

[0012] Specifically, the method for establishing the Zika virus infectious animal model is to obtain a ZIKV-challenged animal model by injecting ZIKV into the tail vein of mice lacking TLR3 or TLR7.

[0013] Furthermore, the samples tested in the ZIKV-challenged animal model are blood, liver, spleen, reproductive organs and brain tissue.

[0014] In a second aspect, the present invention provides a ZIKV challenged animal model obtained by the method.

[0015] 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.

[0016] Compared with the prior art, the present invention is beneficial in that:

[0017] The present invention creates a ZIKV-challenged animal model by inoculating animals lacking Toll-like receptors (TLRs). After ZIKV inoculation, significant ZIKV loads were detected in the animals' blood, liver, spleen, reproductive organs, and brain tissue. Furthermore, the spleen showed significant enlargement, liver tissue showed punctate necrotic lesions, splenic corpuscles were significantly reduced, and mRNA levels of inflammation-related markers such as Ccl5, Cxcl1, Isg15, and Isg56 were significantly upregulated. This indicates that the ZIKV-challenged animal model was successfully constructed and can be used in research related to ZIKV infection mechanisms, vaccine development, and anti-ZIKV drug development. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 These are the ZIKV RT-PCR test results in the liver, spleen, reproductive organs, brain tissue, and blood of TLR3-deficient mice inoculated with ZIKV in Example 1;

[0019] Figure 2 This is a gross photograph of the spleen tissue of TLR3-deficient mice on day 7 after inoculation with ZIKV in Example 1;

[0020] Figure 3 This is the HE staining result of the liver tissue of TLR3-deficient mice on day 12 after inoculation with ZIKV in Example 1;

[0021] Figure 4 These are the results of detecting Ccl5 mRNA levels in the liver, spleen, reproductive organs, and brain tissues of TLR3-deficient mice after inoculation with ZIKV in Example 1;

[0022] Figure 5 The results of Isg15 mRNA level detection in the liver, spleen, reproductive organs, and brain tissues of TLR3-deficient mice after inoculation with ZIKV in Example 1 are as follows;

[0023] Figure 6 These are the ZIKV RT-PCR test results in the liver, spleen, reproductive organs, brain tissue, and blood of TLR7-deficient mice after inoculation with ZIKV in Example 2;

[0024] Figure 7 This is a gross photograph of the spleen tissue of TLR7-deficient mice on day 17 after inoculation with ZIKV in Example 2;

[0025] Figure 8 This is the HE staining result of spleen tissue of TLR7-deficient mice on day 17 after ZIKV inoculation in Example 2;

[0026] Figure 9These are the results of detecting Ccl5 mRNA levels in the liver, spleen, reproductive organs, and brain tissues of TLR7-deficient mice after inoculation with ZIKV in Example 2;

[0027] Figure 10 These are the results of detecting Cxcl1 mRNA levels in the liver, spleen, reproductive organs, and brain tissues of TLR7-deficient mice after inoculation with ZIKV in Example 2;

[0028] Figure 11 The results of Isg15 mRNA level detection in the liver, spleen, reproductive organs, and brain tissues of TLR7-deficient mice after inoculation with ZIKV in Example 2 are as follows;

[0029] Figure 12 These are the results of Isg56 mRNA level detection in the liver, spleen, reproductive organs, and brain tissues of TLR7-deficient mice after inoculation 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 establishing a Zika virus infectious animal model, wherein a ZIKV-challenged animal model is obtained by inoculating ZIKV into a Toll-like receptor (TLR)-deficient animal.

[0032] In some examples, the TLR-deficient animal includes a TLR-deficient mouse, a TLR-deficient rat, a TLR-deficient guinea pig, a TLR-deficient hamster, a TLR-deficient rabbit, a TLR-deficient dog, or a TLR-deficient monkey.

[0033] In some examples, the TLR-deficient animal is a TLR3-deficient animal or a TLR7-deficient animal.

[0034] In some examples, the ZIKV inoculation dose is 1 × 10 4 PFU / each ~1 × 10 5 PFU / mouse.

[0035] In some examples, the vaccination method is intravenous injection; and the intravenous injection is tail vein injection.

[0036] In some examples, the samples examined in the ZIKV-challenged animal model are blood, liver, spleen, reproductive organs, and brain tissue.

[0037] 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.

[0038] In the following specific examples, the ZIKV virus solution preparation method is as follows:

[0039] (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.

[0040] (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.

[0041] (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.

[0042] (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.

[0043] The ZIKV virus titer determination method is as follows:

[0044] (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.

[0045] (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.

[0046] (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.

[0047] (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.

[0048] (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.

[0049] (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.

[0050] (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.

[0051] (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.

[0052] (9) Calculate the virus titer according to the following formula:

[0053] Calculation formula: PFU / mL = average number of plaques / (dilution factor × volume of virus added per well).

[0054] Example 1 TLR3-deficient mouse ZIKV challenge model and effect evaluation

[0055] TLR3 gene-deficient mice were generated at Beijing Weishanglide 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 TLR3 gene-deficient mice were divided into the following groups (D3, D5, D7, and D12, respectively, on days 3, 5, 7, and 12 after virus inoculation):

[0056]

[0057] The ZIKV virus (1×10 5 PFU / mouse), and the control group was injected with the same volume of normal saline.

[0058] At the scheduled sampling time, mice 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 reproductive organs: bilateral testicles were removed from males, and bilateral ovaries were 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 completely, and perform nucleic acid testing on the right lobule. The fifth step was to remove the brain: the skull was cut open along both sides of the occipital bone, and the brain was removed with ophthalmic scissors.

[0059] RT-PCR was used to detect ZIKV viral load in blood, liver, spleen, reproductive organs, and brain tissues to determine viral infection status in these 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 CACGAAGTCT CTATT GC -3'. Results are shown in the table. Figure 1 The figure shows that ZIKV was not detected in the liver tissue of any mouse group, but was detected in the spleen, brain tissue, reproductive organs, and blood. In the early stages of infection, there were no significant differences between the sexes; however, in the later stages of infection, the viral load in males was significantly lower than that in females.

[0060] The spleen tissue of each group of mice was observed. Figure 2 The figure shows that on the 7th day after infection, the spleen of female mice was significantly enlarged, while the external size of the spleen of male mice did not change significantly.

[0061] The organs and tissues of mice in each group were taken for HE staining. The results are shown in Figure 3 The figure shows that on the 12th day after infection, punctate necrotic lesions appeared in the mouse liver tissue, while no obvious pathological changes were observed in other tissues.

[0062] Finally, RT-PCR was used to detect the mRNA levels of inflammation-related molecules in liver, spleen, reproductive organs, and brain tissues, and 1000×2^-△Ct (Ct experimental group-Ct internal reference) was used for relative quantitative multiple calculation. The results are shown in Figure 4 (Ccl5) and Figure 5 The figure shows that the inflammation-related molecules Ccl5 and Isg15 were significantly upregulated in various organs compared with the control group, indicating a significant inflammatory response, indicating successful ZIKV infection.

[0063] Example 2 TLR7-deficient mouse ZIKV challenge model and effect evaluation

[0064] TLR7 gene-deficient mice were generated at Beijing Weishanglide 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 TLR7 gene-deficient mice were divided into the following groups (D4, D9, D13, and D17, respectively, on days 4, 9, 13, and 17 after virus inoculation):

[0065]

[0066] The ZIKV virus (1×10 4 PFU / mouse), and the control group was injected with the same volume of normal saline.

[0067] At the scheduled sampling time, mice 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.

[0068] RT-PCR was used to detect the ZIKV viral load in blood, liver, spleen, genitals, and brain tissues to clarify the viral infection status in the tissues. 1000×2^-△Ct (Ct experimental group - Ct internal reference) was used for relative quantitative multiple calculation. The primers used for ZIKV detection were the same as those in Example 1. The test results are shown in Figure 6 The figure shows that ZIKV was detected in the liver, spleen, genitals, brain tissue and blood samples of mice in each group.

[0069] The spleen tissue of each group of mice was observed. Figure 7 The figure shows that on the 17th day after infection, the mouse spleen was significantly enlarged.

[0070] The organs and tissues of mice in each group were taken for HE staining. The results are shown in Figure 8 The figure shows that on day 17 after infection, the number of splenic bodies in the mouse spleen decreased and lymphohistiocytic proliferation was observed.

[0071] Finally, RT-PCR was used to detect the mRNA levels of inflammation-related molecules in liver, spleen, genitals, and brain tissues, and 2^-△△Ct was used for relative quantitative multiple calculation. The results are shown in Figure 9 (Ccl5), Figure 10 (Cxcl1), Figure 11 (Isg15), Figure 12 The figure shows that the inflammation-related molecules Ccl5, Cxcl1, Isg15, and Isg56 were significantly upregulated in various organs compared with the control group, indicating a significant inflammatory response, indicating successful ZIKV infection.

[0072] In summary, the present invention obtains a ZIKV-challenged animal model by inoculating animals lacking Toll-like receptors (TLRs) with ZIKV. After ZIKV inoculation, significant ZIKV loads were detected in the animals' blood, liver, spleen, reproductive organs, and brain tissue. Furthermore, the spleen showed significant enlargement, punctate necrotic lesions appeared in the liver tissue, splenic corpuscles were significantly reduced, and mRNA levels of inflammation-related indicators Ccl5, Cxcl1, Isg15, and Isg56 were significantly upregulated. This indicates that the ZIKV-challenged animal model of the present invention was successfully constructed and can be used in research related to ZIKV infection mechanisms, vaccine development, and antiviral drug development.

[0073] 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 establishing a Zika virus infectious animal model, characterized in that: By inoculating Toll-like receptor-deficient animals with Zika virus, a Zika virus-challenged animal model was obtained.

2. The method for establishing a Zika virus infectious animal model according to claim 1, characterized in that: The Toll-like receptor-deficient animal is a TLR3-deficient animal or a TLR7-deficient animal.

3. The method for establishing a Zika virus infectious animal model according to claim 1, characterized in that: The Toll-like receptor-deficient animals include: TLR-deficient mice, TLR-deficient rats, TLR-deficient guinea pigs, TLR-deficient hamsters, TLR-deficient rabbits, TLR-deficient dogs or TLR-deficient monkeys.

4. The method for establishing a Zika virus infectious animal model according to claim 1, characterized in that: The inoculation dose of Zika virus is 1 × 10 4 PFU / each ~1 × 10 5 PFU / mouse.

5. The method for establishing a Zika virus infectious animal model according to claim 1, characterized in that: The vaccination method includes but is not limited to intravenous injection.

6. The method for establishing a Zika virus infectious animal model according to claim 1, characterized in that: A Zika virus-challenged animal model was obtained by injecting Zika virus into the tail vein of mice lacking TLR3 or TLR7.

7. A Zika virus challenge animal model established using the method according to any one of claims 1 to 6.

8. Use of the Zika virus-challenged animal model according to claim 7 in the study of Zika virus infection mechanisms, vaccine development, or anti-Zika virus drug development.

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