Construction method of Zika virus challenge animal model

Abstract

The invention discloses a Zika virus ZIKV (Zika virus, ZIKV) challenging animal model construction method, which comprises the following steps: inoculating ZIKV to an F344 rat through an intracranial injection way for modeling to obtain the ZIKV challenging animal model. After inoculation of ZIKV, obvious ZIKV load can be detected in brain tissues, spleens and reproductive organs of F344 rats, and the load of ZIKV is increased along with prolonging of time. It is shown that the ZIKV challenge model is successfully constructed, and the ZIKV challenge model can be applied to related researches of ZIKV infection mechanisms, vaccine research and development, anti-ZIKV drug research and development and the like.

Description

Technical Field

[0001] This invention relates to the field of animal model technology, and in particular to a method for constructing a Zika virus challenge animal model. Background Technology

[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, one for Asia and one for Africa, and it is primarily transmitted through the bite of infected Aedes aegypti mosquitoes. ZIKV was discovered and documented more than 70 years ago. ZIKV infection typically causes mild flu-like symptoms, commonly including fever and rash. Some patients may experience conjunctivitis, muscle and joint pain, and general weakness. Due to its high neurotropism, ZIKV infection during pregnancy may lead to microcephaly in newborns, while adult infection may cause Guillain-Barré syndrome. Currently, there are no safe and effective vaccines or specific anti-ZIKV drugs to prevent Zika virus infection.

[0003] Constructing effective Zika virus challenge animal models is of great significance for studying the Zika virus infection mechanism and developing specific anti-ZIKV drugs. Therefore, to overcome the shortcomings of existing technologies, such as long modeling time, low model stability, and high cost, it is necessary to develop a method for constructing a Zika virus challenge animal model. Summary of the Invention

[0004] The purpose of this invention is to provide a method for constructing a ZIKV challenge animal model, which has the advantages of simple operation, short modeling time, high model stability, and low cost. The ZIKV challenge model has been successfully constructed and can be used in research on ZIKV infection mechanism, vaccine development, and anti-ZIKV drug development.

[0005] To achieve the aforementioned objective, the present invention adopts the following technical solution: In a first aspect of the present invention, a method for constructing a ZIKV challenge animal model is provided, the method comprising: A ZIKV challenge animal model was obtained by inoculating F344 rats with ZIKV via intracranial injection.

[0006] Furthermore, the inoculation dose of ZIKV is 1×10⁻⁶. 5 PFU / each.

[0007] Furthermore, the ZIKV strain is the wild-type Zika virus strain SZ-WIV01 with Genebank accession number KU963796.

[0008] Furthermore, the molding time is 7-28 days.

[0009] Furthermore, the method also includes: Collect the samples to be tested and evaluate the model.

[0010] Furthermore, the tested samples include the spleen, reproductive organs, and brain tissue. Brain tissue is preferably the tested sample from the ZIKV challenge animal model.

[0011] In a second aspect of the invention, the application of the ZIKV challenge animal model constructed by the method is provided in the preparation of reagents for studying the ZIKV infection mechanism.

[0012] In a third aspect of the invention, the application of the ZIKV challenge animal model constructed by the method is provided in the preparation of a platform for evaluating ZIKV infection mechanism vaccines.

[0013] In a fourth aspect of the invention, the application of the ZIKV challenge animal model constructed by the method is provided in a drug screening system for preparing drugs against ZIKV infection.

[0014] In a fifth aspect of the invention, the application of the ZIKV challenge animal model constructed by the method is provided in evaluating the pathogenicity of anti-ZIKV for non-diagnostic purposes.

[0015] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages: This invention provides a method for constructing a ZIKV challenge animal model. The method involves inoculating F344 rats with ZIKV to obtain the ZIKV challenge animal model. After ZIKV inoculation, significant ZIKV loads were detected in the brain tissue, spleen, and reproductive organs of the rats, and these loads increased over time. These results fully demonstrate that the ZIKV-infected F344 rat challenge model of this invention was successfully constructed and can serve as a novel and highly effective animal model for low-pathogenicity viruses, applicable to research on ZIKV infection mechanisms, vaccine development, and anti-ZIKV drug development. This invention establishes the first F344 rat ZIKV infection model by optimizing the intracranial injection route (1×10⁻⁶). 5 PFU / each) achieves: (1) It breaks through the blood-brain barrier and directly targets the central nervous system. (2) The virus replicates efficiently in brain tissue (the viral load increases 3.8 times after 4 weeks of infection).

[0016] (3) Simultaneous infection of the spleen and reproductive organs to simulate the pathological features of multiple organs. Attached Figure Description

[0017] Figure 1Results of ZIKV mRNA level detection in brain tissue of F344 rats at different time points after ZIKV injection; Figure 2 Results of ZIKV mRNA level detection in spleen tissue of F344 rats at different time points after ZIKV injection; Figure 3 Results of ZIKV mRNA level detection in reproductive tissues of F344 rats at different time points after ZIKV injection. Detailed Implementation

[0018] The following detailed description of the embodiments and examples will illustrate the present invention in more detail, thereby making the advantages and various effects of the embodiments more clearly apparent. Those skilled in the art should understand that these detailed embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0019] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the embodiments of this invention pertain. In the event of any conflict, this specification shall prevail.

[0020] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in the embodiments of the present invention can be obtained by purchasing them on the market or by existing methods.

[0021] The present application will now be described in detail with reference to embodiments and experimental data.

[0022] Example 1: Method for constructing an animal model of Zika virus challenge I. Preparation of ZIKV viral fluid 1. The method for preparing the ZIKV viral fluid is as follows: (1) Wild-type Zika virus strain SZ-WIV01 with Genebank accession number KU963796. After the strain was thawed naturally, it was inoculated into a T75 culture flask containing C636 cells with a growth density of about 80% (cultured in DMEM medium (Gibco) containing 2% fetal bovine serum (Meilune)) and cultured for 3 days in a 27 ℃, 5% CO2 incubator (Memmert, INCO153).

[0023] (2) After microscopic examination reveals that half of the cells are floating, harvest them. First, collect the supernatant in the culture flask into a 50 mL centrifuge tube and centrifuge at 3000 rmg and 4 ℃ for 10 min. Leave about 2 mL of liquid in each culture flask, scrape the cells from the culture flask with a cell scraper and put them into another 15 mL centrifuge tube.

[0024] (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, and store it at 4 ℃ for later use; blow away the cell pellet after centrifugation with 15 mL of cell solution, aspirate it into a 15 mL centrifuge tube, freeze it at -80 ℃ or in liquid nitrogen, and then heat it in a 37 ℃ water bath to dissolve it. Repeat this process 3 times.

[0025] (4) Place the frozen and thawed cell solution into a centrifuge and centrifuge at 3000 rmg and 4 ℃ for 10 min. Aspirate the supernatant with a syringe and add it to the previously filtered virus solution. Mix well and divide 3 mL into 3 vials for plaque experiments. Then transfer to a -80 ℃ freezer for storage.

[0026] 2. The method for determining the ZIKV virus titer is as follows: (1) Vero E6 cells were cultured at 37 ℃ in a 5% CO2 incubator until the growth density reached about 90%. The culture medium was 1% methylcellulose medium. The medium was then aspirated and discarded, and 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 they detached, with a maximum digestion time of 5 min.

[0027] (2) After digestion, first wash the adherent cells off the culture flask with 2 mL of DMEM medium (with 10% fetal bovine serum and 1% antibiotics), and then repeatedly pipette the cells until there is no obvious clumping. Then add 10 mL of culture medium, tighten the cap, and shake the culture flask to mix the cells evenly.

[0028] (3) Add the mixed cell suspension to each well of a 24-well plate, adding 0.5 mL to each well. Incubate overnight in a CO2 incubator and observe the cells under a microscope. Proceed to the next step once the cells have adhered to the plate and filled each well.

[0029] (4) Prepare 6 EP tubes. Add 900 μL of DMEM to each EP tube. Add 100 μL of the virus stock solution to the first EP tube, then mix it thoroughly by pipetting. Add 100 μL to the second EP tube. Repeat the above steps until all 6 EP tubes are prepared. The result is obtained from 10... -1 Up to 10 -6 Six concentrations of virus solution.

[0030] (5) Transfer the culture plate from the incubator to the biosafety cabinet, aspirate the culture medium from the culture plate, and then add 500 μL of virus solution to four wells for each concentration. After completion, put the culture plate back into the incubator and allow it to adsorb for 1-2 h.

[0031] (6) After adsorption is complete, aspirate and discard the supernatant, then rinse 2-3 times with DMEM. Then add 500 μL of 1% methylcellulose medium to each well. Then return to the incubator and incubate for 7 days.

[0032] (7) After the culture is completed, aspirate and discard the culture medium, then rinse with DMEM 2-3 times, and add 0.5-1 mL of fixative to fix for more than 30 min. After fixation, aspirate and discard the fixative, and add crystal violet staining solution for 10 min. Then rinse away the excess staining solution slowly with running water.

[0033] (8) Calculate the number of empty spots in the four holes that are visible by visual inspection at the smallest magnification, and then take the average value of the four holes.

[0034] (9) Calculate the viral titer using the following formula: Calculation formula: PFU / mL = Average number of plaques / (Dilution factor × Volume of virus added per well).

[0035] II. Model Construction F344 rats were bred and produced by the Animal Experiment Center of Wuhan University. The animal experimental projects and protocols were reviewed and approved by the Experimental Animal Husbandry and Management Committee (IACUC) of the Animal Experiment Center of Wuhan University before implementation. All experiments were conducted in the ABSL-2 laboratory. Three-week-old male F344 rats were divided into two groups: a 1-week injection group and a 4-week injection group, with four rats in each group. ZIKV (1×10⁻⁶) was administered intracranially. 5 PFU / each).

[0036] Example 2, Model Evaluation At 1 week and 4 weeks after injection, rats were euthanized by CO2 asphyxiation. After death by asphyxiation, bilateral testes, liver, spleen and brain were removed.

[0037] RT-PCR was used to detect ZIKV mRNA levels in the brain, testes, spleen, and liver to evaluate viral load and determine the viral infection status in the tissues. The relative quantification fold was calculated using 1000 × 2 - ΔCt (ZIKV Ct - internal control Ct). The primers used for ZIKV detection were: F: 5'- TTGGT CATGA TACTG CTGAT TGC -3'; R: 5'- CCCTC CACGAAGTCT CTATT GC-3'.

[0038] The results of ZIKV mRNA detection in brain tissue are as follows: Figure 1As shown, ZIKV mRNA signals were detectable in brain tissue 1 week post-infection, and the ZIKV mRNA level significantly increased by 4 weeks post-infection. The ZIKV mRNA level in brain tissue was also significantly higher than in other tissues. This indicates that ZIKV replicates in brain tissue, indicating successful ZIKV infection, and that brain tissue is the optimal organ for examination.

[0039] The results of ZIKV mRNA detection in spleen tissue are as follows: Figure 2 As shown, consistent with the detection results in brain tissue, ZIKV mRNA signals could be detected 1 week after infection, and the ZIKV mRNA level increased significantly by 4 weeks after infection. This indicates that ZIKV also replicates in spleen tissue, and ZIKV infection was successful.

[0040] The results of ZIKV mRNA detection in reproductive tissues are as follows: Figure 3 As shown, consistent with the detection results in brain and spleen tissues, the mRNA level of ZIKV in reproductive tissues increased over time after ZIKV injection. This indicates that ZIKV replicates in reproductive tissues, and ZIKV infection was successful.

[0041] In summary, this invention establishes a ZIKV challenge animal model by inoculating F344 rats with ZIKV. Following ZIKV inoculation, significant ZIKV loads were detected in the spleen, reproductive organs, and brain tissue of the rats. Furthermore, the ZIKV load increased with prolonged infection time, indicating that the ZIKV challenge model of this invention was successfully constructed and can be used for research related to ZIKV infection mechanisms, vaccine development, and anti-ZIKV drug development.

[0042] Finally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0043] Although preferred embodiments of the present invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the embodiments of the present invention.

[0044] Obviously, those skilled in the art can make various modifications and variations to the embodiments of the present invention without departing from the spirit and scope of the embodiments of the present invention. Therefore, if these modifications and variations to the embodiments of the present invention fall within the scope of the claims of the embodiments of the present invention and their equivalents, the embodiments of the present invention are also intended to include these modifications and variations.

Claims

1. A method for constructing an animal model of Zika virus challenge, characterized in that, The method includes: A Zika virus challenge animal model was obtained by inoculating F344 rats with Zika virus via intracranial injection.

2. The method for constructing a Zika virus challenge animal model according to claim 1, characterized in that, The inoculation dose of the Zika virus is 1×10⁻⁶. 5 PFU / each.

3. The method for constructing a Zika virus challenge animal model according to claim 1, characterized in that, The Zika virus strain in question is the wild-type Zika virus strain SZ-WIV01, with Genebank accession number KU963796.

4. The method for constructing a Zika virus challenge animal model according to claim 1, characterized in that, The F344 rats were male individuals aged 3-7 weeks.

5. The method for constructing a Zika virus challenge animal model according to claim 1, characterized in that, The method also includes: collecting the tested samples and testing the evaluation model.

6. The method for constructing a Zika virus challenge animal model according to claim 5, characterized in that, The samples examined included the spleen, reproductive organs, and brain tissue.

7. The application of the Zika virus challenge animal model constructed by any of the methods in claims 1-6 in the preparation of reagents for studying the Zika virus infection mechanism.

8. The application of the Zika virus challenge animal model constructed by any of the methods in claims 1-6 in the preparation of a Zika virus infection mechanism vaccine evaluation platform.

9. The application of the Zika virus challenge animal model constructed by any of the methods in claims 1-6 in the preparation of a drug screening system for Zika virus infection.

10. The application of the Zika virus challenge animal model constructed by any of the methods in claims 1-6 in evaluating the pathogenicity of Zika virus for non-diagnostic purposes.

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