Construction method and application of animal model of hepatitis e virus infection

Abstract

The application discloses a construction method and application of an animal model infected with hepatitis E virus. Specifically disclosed is a construction method of an animal model infected with hepatitis E virus, which comprises the following steps: encapsulating complete genome RNA of hepatitis E virus in LNP to obtain LNP-encapsulated hepatitis E virus genome RNA; inoculating the LNP-encapsulated hepatitis E virus genome RNA into a non-human mammal to obtain an animal model infected with hepatitis E virus. The RNA dosage is as low as 1 ug, and the animal infection model can be established. The application solves the technical barrier that the hepatitis E virus is not suitable for in-vitro culture, and the virus does not need to be isolated from nature or clinical specimens. The LNP-encapsulated HEV genome RNA can be obtained through gene synthesis, and is used as an inoculation material of the animal infection model, so that the LNP-encapsulated HEV genome RNA is convenient to obtain, has good uniformity and repeatability, and is safer to operate.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a method for constructing an animal model of hepatitis E virus infection and application thereof. BACKGROUND

[0002] Hepatitis E virus (HEV) belongs to the family of Hepeviridae and is a single-stranded positive-sense RNA virus, which is transmitted through the fecal-oral route and is one of the main pathogens causing acute and chronic hepatitis. In HEV high prevalence areas, the total incidence of hepatitis E is 1%-15%. Most of the clinical symptoms of hepatitis E are self-limiting acute infection, however, the rate of pregnant women infected with HEV developing fulminant hepatitis (acute liver failure) is high, and the mortality rate is close to 30%. In addition, HEV can cause persistent infection in organ transplant patients and immunodeficient patients, and this HEV-induced chronic hepatitis has become an important clinical problem. Due to the lack of efficient animal infection models and in vitro cell culture systems, the research on the pathogenic mechanism, vaccine and antiviral drugs of HEV has been greatly limited.

[0003] HEV is extremely difficult to culture in vitro, although a few studies have reported that HEV wild strains can be cultured in vitro, but it is difficult to repeat. Therefore, the inoculum for the construction of the commonly used HEV infection animal model is mostly fecal suspension or serum from infected animals, that is, the infected sample is directly injected. However, the infected sample is not renewable, even if the virus can be obtained by continuous passage in animals, there is still a problem of viral genome mutation leading to virulence decline over time, and the uniformity cannot be guaranteed. Therefore, obtaining a stable virus seed solution is the bottleneck for the construction of HEV infection animal models. In recent years, there have been reports that in vitro transcribed RNA of HEV can be used for liver injection in animals, which can reproduce the characteristics of HEV infection in vivo. However, liver injection is traumatic, and the infection efficiency of this method is low. Therefore, it is urgent to develop a rapid, cell-free, and less traumatic HEV infection animal model.

[0004] Lipid nanoparticle (LNP) is an effective delivery system, which has great potential in mRNA vaccines, gene editing and cancer treatment. LNP delivery system can prevent rapid degradation of RNA in vivo, and at the same time can greatly reduce the natural immune response of the host to exogenous RNA. So far, there has been no report on the establishment of an infected animal model by intravenous delivery of viral genomic RNA based on LNP technology. SUMMARY

[0005] The technical problem to be solved by the present application is how to construct an animal model infected with hepatitis E virus. The technical problem to be solved is not limited to the technical subject as described, and other technical subjects not mentioned herein can be clearly understood by those skilled in the art through the following description.

[0006] To solve the above technical problem, the present application first provides a method for constructing an animal model infected with hepatitis E virus, which can include encapsulating a complete genome RNA of hepatitis E virus in LNP to obtain LNP-encapsulated hepatitis E virus genome RNA, inoculating the LNP-encapsulated hepatitis E virus genome RNA into a non-human mammal to obtain the animal model infected with hepatitis E virus.

[0007] In the above construction method, the inoculation can be intravenous injection.

[0008] In the above construction method, the structure of the complete genome RNA of hepatitis E virus can include a 5' non-coding region, a coding region, and a 3' non-coding region.

[0009] In the above construction method, the non-human mammal can be selected from any one of a rabbit, a mouse, and a monkey.

[0010] In the above construction method, the non-human mammal can be a rabbit.

[0011] Further, the encapsulation includes a step of mixing the complete genome RNA of hepatitis E virus with a lipid excipient composition, the lipid excipient composition including a cationic lipid, distearoyl phosphatidylcholine, cholesterol, and PEG-lipid.

[0012] Further, the molar ratio of the cationic lipid, distearoyl phosphatidylcholine, cholesterol, and PEG-lipid in the lipid excipient composition can be, but is not limited to, 50:10:38:1.5.

[0013] Further, the mixing can be mixing the complete genome RNA of hepatitis E virus with the lipid excipient composition at a volume ratio of 1:2.

[0014] The cationic lipid described herein can include DLin-MC3-DMA, ALC-0315, SM-102, etc., but is not limited thereto. Specifically, in an embodiment of the present application, the cationic lipid is DLin-MC3-DMA.

[0015] The PEG-lipid described herein can include PEG 2000C-DMG, ALC-0159, PEG-DMG, PEG-DSPE, PEG-DSG, PEG-dipalmitoyl, PEG-dioleoyl, PEG-distearyl, PEG-DAG, PEG-DPPE, PEG-PE, PEG-S-DAG, PEG-cer, and the like, but are not limited thereto. Specifically, in one embodiment of the present application, the PEG-lipid is PEG 2000 C-DMG.

[0016] The lipid adjuvant composition described herein can be a solution comprising the cationic lipid, distearoylphosphatidylcholine, cholesterol, and PEG-lipid dissolved in a solvent, which can be ethanol.

[0017] The present application also provides a method for constructing an animal model of hepatitis E virus infection, which can comprise the following steps:

[0018] A1) linearizing the recombinant vector PUC57-R14 with Hind III enzyme, and transcribing the viral genomic RNA in vitro after purification; the structure of the viral genomic RNA comprises a 5' non-coding region, a coding region, and a 3' non-coding region; the nucleotide sequence of the recombinant vector PUC57-R14 is SEQ ID No. 1;

[0019] A2) mixing the viral genomic RNA with a lipid adjuvant composition, encapsulating at room temperature, dialyzing in PBS solution at 4°C overnight, and then concentrating and filtering to sterilize to obtain LNP-encapsulated hepatitis E virus RNA; the lipid adjuvant composition comprises a cationic lipid, distearoylphosphatidylcholine, cholesterol, and PEG-lipid;

[0020] A3) inoculating a non-human mammal with the LNP-encapsulated hepatitis E virus RNA by intravenous injection;

[0021] A4) extracting viral nucleic acid from a fecal suspension or tissue homogenate liquid sample of the inoculated non-human mammal for real-time fluorescent quantitative PCR detection, and obtaining the animal model of hepatitis E virus infection when hepatitis E virus RNA is present in the sample.

[0022] In the above construction method, the non-human mammal can be selected from any one of a rabbit, a mouse, and a monkey.

[0023] In the above construction method, the non-human mammal can be a rabbit.

[0024] In the above construction method, the molar ratio of the cationic lipid, distearoylphosphatidylcholine, cholesterol, and PEG-lipid in the lipid adjuvant composition can be 50:10:38:1.5.

[0025] In the above construction method, the cationic lipid can be specifically DLin-MC3-DMA, and the PEG-lipid can be specifically PEG-DMG. 2000 -C-DMG.

[0026] The present application also provides any one of the following uses of the animal model of hepatitis E virus infection constructed by any one of the construction methods described herein:

[0027] B1) use in screening anti-hepatitis E virus drugs or hepatitis E vaccines;

[0028] B2) use in evaluating the therapeutic effect of anti-hepatitis E virus drugs or the effect of hepatitis E vaccines;

[0029] B3) use in functional research on key regions or sites of the hepatitis E virus genome;

[0030] B4) use in research on the pathogenesis of hepatitis E virus infection.

[0031] The present application also provides an animal model of hepatitis E virus infection, which can be constructed by any one of the construction methods described herein.

[0032] The present application discloses a synthetic LNP hepatitis E virus RNA (LNP-R14) obtained by encapsulating hepatitis E virus RNA viral genome with lipid nanoparticles (LNP), and a method for directly infecting animals with the synthetic LNP hepatitis E virus RNA, which can make the animals exhibit typical viral infection characteristics, and an RNA amount as low as 1 ug can establish an animal infection model. After infection, viral infection characteristics are identified by monitoring fecal virus excretion, urine antigen, liver viral load, and liver pathological analysis. The animal model can be applied to the evaluation of anti-HEV drugs and vaccines. The present application also discloses a use of the above animal infection model for in vivo evaluation of hepatitis E virus vaccines and antiviral drugs. The present application has the following advantages: (1) it solves the technical barrier of hepatitis E virus which is not suitable for in vitro culture; (2) it does not need to isolate viruses from nature or clinical specimens, and can obtain LNP-encapsulated HEV genomic RNA by gene synthesis, which is convenient to obtain and has good uniformity and repeatability. Compared with viral strains, viral genomic RNA is safer to operate; (3) the synthetic LNP hepatitis E virus RNA can be directly genetically modified, and the function of key regions or sites of the viral genome can be studied in the animal infection model. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 Results of in vitro transcription experiment in Example 1.

[0034] Figure 2 The results of R14 LNP particle size detection in Example 1 are shown.

[0035] Figure 3 This is a schematic diagram of the overall experimental process in Example 2.

[0036] Figure 4 This is a schematic diagram of the grouping and operation of the animal infection experiment in Example 2.

[0037] Figure 5 This is an example of the changes in HEV RNA levels in the feces of rabbits in each group throughout the entire infection process in Example 2.

[0038] Figure 6 This is an example of the changes in HEV ORF2 antigen levels in the urine of rabbits in each group throughout the entire infection process in Example 2.

[0039] Figure 7 Example 2: HEV in different tissues and organs of rabbits in different groups on day 21 post-infection. RNA levels.

[0040] Figure 8 The results of HE staining of rabbit liver tissue from each group in Example 2 are shown.

[0041] Figure 9 Example 3 shows the design and flowchart for evaluating the efficacy of antiviral drugs.

[0042] Figure 10 Example 3: Evaluation of the effect of antiviral drugs on rabbit fecal HEV RNA levels. Detailed Implementation

[0043] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0044] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0045] Example 1: Encapsulation of HEV viral genomic RNA LNP

[0046] 1. In vitro transcription of HEV strain R14 genomic RNA

[0047] Linearization of R14 infectious clone vector with Hind III enzyme, reaction system (100ul) as shown in Table 1. R14 plasmid (R14 infectious clone vector PUC57-R14) sequence as shown in SEQ ID No. 1.

[0048] Table 1, R14 plasmid linearization reaction system

[0049] Reagent Amount Buffer (10x) 10ul PUC57-R14 100 ug / 10 ul Hind III 10ul H2O 70ul

[0050] The linearized plasmid was purified by phenol chloroform extraction. The specific steps are as follows: (1) add 200ul of H2O to the enzyme digestion product, and then add 300ul of DNA extraction solution (phenol: chloroform: isoamyl alcohol = 25:24:1); (2) centrifuge at 13000rpm for 10min, and retain the supernatant; (3) add an equal volume (300ul) of nucleic acid extraction solution (chloroform: isoamyl alcohol = 24:1), centrifuge for 5min, and retain the supernatant; (4) add 750ul of anhydrous ethanol, precipitate at -80℃ for 30min, centrifuge at 4℃ at 13000rpm for 10min, and retain the precipitate; (5) wash once with 1ml of 75% ethanol, centrifuge at 13000rpm for 5min; (6) air dry the precipitate, and dissolve with 50ul of RNase free H2O; (7) measure the purified DNA concentration using nanodrop.

[0051] In vitro transcription was performed using linearized DNA, 37℃ reaction for 6hr, reaction system (30ul system) as shown in Table 2 (in vitro transcription reagent purchased from Promega, item number: P1300). The in vitro transcribed RNA can be detected by agarose electrophoresis, and the results are as shown in Figure 1 , R14 from top to bottom two bands, the first is the DNA template, and the second is the in vitro transcribed RNA.

[0052] Table 2, in vitro transcription reaction system

[0053] 5x Buffer 6ul rNTP 6 ul (A:C:U:G=3:3:3:1 mix) Cap 2ul T7 enzymix 2.5ul Linearized DNA 1 ug ddH2O 14.5ul

[0054] 2, HEV strain R14 genomic RNA LNP encapsulation

[0055] Respectively, 30ug of R14 genomic RNA obtained by in vitro transcription was dissolved in 20mM citric acid buffer (PH = 4.0), and then combined with the lipid excipient composition (cationic lipid (DLin-MC3-DMA), distearoyl phosphatidylcholine, cholesterol and PEG-lipid (PEG 2000C-DMG) were dissolved in ethanol at a ratio of 50:10:38:1.5 (molar ratio) at room temperature, and the R14 genomic RNA was encapsulated in the lipid nanosphere preparation by rapid mixing at a volume ratio of 1:2. After dialysis in a PBS solution (purchased from Beijing Solabio Technology Co., Ltd., item number P1020) at 4°C overnight, the LNP-R14 preparation was obtained by concentration and filtration sterilization. Thus, the LNP-encapsulated hepatitis E virus genomic RNA was synthesized and named LNP-R14. The corresponding particle size detection results are shown in Figure 2 The diameter of LNP-R14 was about 100 nm.

[0056] Example 2: Verification of the infectivity of LNP-R14 in a rabbit model

[0057] Using the LNP-R14 prepared in Example 1, a hepatitis E virus-infected animal model, i.e., a LNP-R14-infected rabbit (also referred to as a LNP-R14 rabbit model), was constructed, and the constructed LNP-R14-infected rabbit was identified. The overall experimental flowchart for constructing the animal model is shown in Figure 3

[0058] 1. Experimental animal enrollment and screening

[0059] The Japanese large-ear white rabbits (2.5-3.5 months old, half male and half female, and weighing about 2.5-3 kg) used in the study were purchased from Beijing Jinmuyang Experimental Breeding Co., Ltd. All the experimental rabbits were housed in cages and given sufficient water and feed, and the experimental animals had passed the experimental animal ethics review by the Animal Ethics Committee of the Medical Department of Peking University before the experiment.

[0060] Before the experiment, the serum and fecal samples of all the experimental rabbits were collected, the HEV RNA in the feces was detected by RT-qPCR, and the anti-HEV antibodies in the serum were detected by enzyme-linked immunosorbent assay (ELISA), and the rabbits with negative anti-HEV antibodies and HEV RNA were enrolled.

[0061] 2. Experimental design and sample collection

[0062] The nanoparticle-coated HEV RNA (LNP-R14) was inoculated into the large-ear white rabbits by intravenous injection, and the injection method is shown in Table 3 and Figure 4

[0063] Table 3: Experimental grouping and inoculation dose

[0064]

[0065]

[0066] ​​Following viral inoculation, urine, blood, and fecal samples were collected weekly for the detection of virological indicators such as fecal HEV RNA, urinary HEV antigen, and serum anti-HEV antibody, with continuous monitoring. Fecal samples were prepared into a 20% (wt / vol) fecal suspension using sterile PBS (pH 7.4), centrifuged at 4°C and 5000 rpm for 20 min, and the supernatant was used for further experiments or stored at -80°C for later use. Blood samples were centrifuged at 4°C and 5000 rpm for 20 min to obtain serum, which was used for further experiments or stored at -80°C for later use. At week 3 (21 dpi) post-viral inoculation, four rabbits from each group were euthanized and dissected. After dissection, the liver, kidneys, duodenum, urine, and bile were collected; the heart and brain were collected from one rabbit from each group. Tissue samples were homogenized to 10% (wt / vol). 0.1g of tissue was placed in a 1.5mL EP tube, and 1mL of pre-chilled PBS and zirconia grinding beads were added. The homogenizer was then used with an automated tissue homogenizer at 4℃, 60Hz, and 60s for 5 cycles, with a 9s interval between each cycle. After homogenization, the sample was centrifuged at 8000rpm for 5min. The supernatant was collected for analysis, and the remaining supernatant was transferred to a clean EP tube and stored at -80℃ for later use. Another tissue sample was fixed in 10% formalin for 24h and sent to Seville Biotechnology Co., Ltd. for HE staining. The HE results are as follows: Figure 8 As shown.

[0067] The results suggest that high-dose LNP inoculation can cause significant inflammatory cell infiltration and other damage in rabbit liver tissue.

[0068] 3. HEV RNA extraction and detection

[0069] 3-1. Extraction of viral nucleic acid from liquid specimens:

[0070] Strictly in accordance with The Viral DNA / RNA Kit (AllGold) requires the extraction of viral nucleic acid from liquid samples such as fecal suspension, tissue homogenate, and bile. Detailed steps are as follows:

[0071] 1) Add 20 μL of Proteinase K to a sterile 1.5 mL EP tube without RNase treatment, then add 200 μL of BB5 and vortex to mix for 15 seconds (for multiple samples, Proteinase K and BB5 can be mixed at a ratio of 1:10 and aliquoted into 220 μL tubes for use).

[0072] 2) Add 200 μL of sample to the EP tube, vortex to mix for 15 s, and then incubate in a 56 ℃ water bath for 15 min.

[0073] 3) Remove the EP tube, wipe off the liquid outside the tube wall with absorbent paper and remove it instantly. Add 250 μL of anhydrous ethanol into the EP tube, vortex and mix for 15 seconds, and let it stand at room temperature for 5 minutes.

[0074] 4) Add the solution to the centrifuge column (place the centrifuge column inside the collection tube), centrifuge for 1 min at room temperature and 12000g, and discard the effluent in the collection tube.

[0075] 5) Add 500 μL of WB5 to the centrifuge column (make sure anhydrous ethanol has been added before use), centrifuge at room temperature and 12000g for 1 min, and discard the effluent in the collection tube.

[0076] 6) Repeat step 5) once.

[0077] 7) Centrifuge at room temperature and 12000g for 1 min to remove residual WB5, so that residual WB5 will not inhibit subsequent reactions.

[0078] 8) Transfer the centrifuge column into a new sterile 1.5 mL EP tube that has been treated with RNase-free, and add 20 μL of RNase-free water to the center of the centrifuge column. Incubate at room temperature for 1 min.

[0079] 9) Centrifuge at room temperature and 12000g for 1 min to elute RNA and obtain approximately 20 μL of RNA.

[0080] 10) Perform reverse transcription of the RNA immediately or store it in a -80°C freezer for later use.

[0081] 3-2. Real-time quantitative PCR detection of viral RNA copy number

[0082] (1) Primers and probes

[0083] Real-time quantitative PCR (RT-qPCR) using reverse transcription employs a TaqMan probe detection method, where the 3' end of the probe is labeled with BHQ-1 and the 5' end with 6-FAM. Specific information on the primers and probes is shown in Table 4.

[0084] Table 4. Primers and probes for RT-qPCR reaction

[0085]

[0086] (2) RT-qPCR

[0087] Strictly in accordance with Follow the instructions to prepare the Probe 1-Step RT-qPCR System Kit (Promega, catalog number A6121) and set the reaction parameters. Prepare 20 μL of the kit for each sample, with two independent replicates per sample. After loading, insert the kit into a Roche container. In 480Ⅱ, reaction parameters were set. After the reaction, negative and positive controls were used to ensure the reliability of the experimental results. Based on the CP values ​​of the samples and standard plasmids calculated by the instrument, the copy numbers of the two standard plasmids with known copy numbers were sequentially matched with the copy numbers of the standard curve. The instrument automatically calculated the copy number of the sample based on the sample CP value, recorded the copy number of the two replicates, calculated the average value, and converted it into the HEV copy number of the corresponding sample. The RT-qPCR reaction system is shown in Table 5.

[0088] Table 5. RT-qPCR reaction system

[0089] Reagent Amount 2x Go-Taq Probe qPCR Master Mix 10.0 μL 50x GoScript RT Mix for 1-step RT-qPCR 0.4 μL JVHEVF primer (10 μmol / L) 1.0 μL JVHEVR-rab primer (10 μmol / L) 1.0 μL Probe (10 μmol / L) 0.5 μL RNase-free water 5.1 μL Total RNA 2.0 μL Total volume 20.0 μL

[0090] RT-qPCR parameters: 45℃ for 30 min, 95℃ for 15 min; 95℃ for 10 s, 55℃ for 20 s, 72℃ for 15 s, 40 cycles; 37℃ for 2 min.

[0091] Quantitative results of fecal suspension are as follows Figure 5 As shown, within three weeks of LNP-R14 inoculation, all eight rabbits in the 100μg and 10μg groups showed fecal viral shedding, only one rabbit in the 1μg group did not shed the virus, and no fecal viral shedding was detected in the 0.1μg group. This demonstrates that LNP-R14 can effectively establish infection and fecal viral shedding in the rabbit model.

[0092] Quantitative results of rabbit tissue as follows Figure 7 As shown, in the 100 μg group, all sacrificed rabbits showed HEV RNA positivity in their liver, kidney, small intestine, heart, and bile, while some rabbit tissues in the 10 μg and 1 μg groups were nucleic acid positive. This indicates that HEV replication can occur in various extrahepatic tissues in rabbits after inoculation with LNP-R14.

[0093] 4. HEV antigen detection

[0094] Urine samples were tested using the ELISA double-antibody sandwich method. The experiment was conducted strictly according to the instructions of the Hepatitis E Virus Antigen Detection Kit (Beijing Wantai, Medical Device Registration Certificate No.: 20163402326). The specific steps are as follows:

[0095] 1) Solution preparation: Dilute 50mL of 20× concentrated washing solution with deionized water to 1L for later use.

[0096] 2) Numbering: Number the microwells corresponding to each sample in sequence, and set up 3 negative control wells and 2 positive control wells.

[0097] 3) Sample addition: Add 100 μL of the test sample, standard, blank, negative or positive control to each well.

[0098] 4) Incubation: After sealing the plate with sealing film, place it in a 37℃ incubator for 1 hour.

[0099] 5) Washing: Wash the board 5 times with a board washing machine, and then dry it.

[0100] 6) Add enzyme: Add 100 μL of enzyme-labeled reagent to each well.

[0101] 7) Incubation: After sealing the plate with sealing film, place it in a 37℃ incubator for 30 minutes.

[0102] 8) Washing: Same as step 5).

[0103] 9) Color development: Add 50 μL of color developer A and B to each well, seal the plate with the sealing film, and place it in a 37°C incubator for 15 min.

[0104] 10) Measurement: Add 50 μL of stop solution to each well, set the parameters of the microplate reader to dual wavelengths 450 / 630 nm, and measure the absorbance (A) value of each well.

[0105] 11) Result Interpretation: Critical value = 0.12 + mean value of negative control well A. If sample A value > critical value, it is judged as positive; if sample A value ≤ critical value, it is judged as negative. S / CO (signal to cutoff) value calculation: sample A value / critical value. Similarly, if S / CO value > 1, it is judged as positive; if S / CO value ≤ 1, it is judged as negative.

[0106] Antigen test results as follows Figure 6 As shown, on day 21 post-infection, all eight rabbits in the 100 μg group tested positive for urine antigens, some rabbits in the 10 μg and 1 μg groups tested positive for urine antigens, and all rabbits in the 0.1 μg group tested negative for urine antigens. The results indicate that LNP-R14 infection in rabbits can lead to dose-related urinary antigen positivity.

[0107] 5. Detection of anti-HEV antibodies

[0108] Serum samples were tested for anti-HEV total antibody using the ELISA double-antigen sandwich method. The procedure was strictly followed according to the instructions for the Hepatitis E Virus Antibody Detection Kit (Beijing Wantai, batch number: ES20230201A). The specific steps are as follows:

[0109] 1) Solution preparation: Dilute 50mL of 20× concentrated washing solution with deionized water to 1L for later use.

[0110] 2) Numbering: Number the microwells corresponding to each sample in sequence, and set up 3 negative control wells and 2 positive control wells.

[0111] 3) Dilution and sample addition: Add 50 μL of sample diluent to each well, followed by 50 μL of the test sample and negative or positive control.

[0112] 4) Incubation: After sealing the plate with sealing film, place it in a 37℃ incubator for 30 minutes.

[0113] 5) Washing: Wash the board 5 times with a board washing machine, and then dry it.

[0114] 6) Add enzyme: Add 100 μL of enzyme-labeled reagent to each well.

[0115] 7) Incubation: After sealing the plate with sealing film, place it in a 37℃ incubator for 30 minutes.

[0116] 8) Washing: Same as step 5).

[0117] 9) Color development: Add 50 μL of color developer A and B to each well, seal the plate with the sealing film, and place it in a 37°C incubator for 15 min.

[0118] 10) Measurement: Add 50 μL of stop solution to each well, set the parameters of the microplate reader to dual wavelengths 450 / 630 nm, and measure the absorbance (A) value of each well.

[0119] 11) Result Interpretation: Critical value = 0.12 + mean value of negative control A. If sample A value > critical value, it is judged as positive; if sample A value ≤ critical value, it is judged as negative. S / CO value calculation: sample A value / critical value. Similarly, if S / CO value > 1, it is judged as positive; if S / CO value ≤ 1, it is judged as negative.

[0120] Antibody test results proved that the rabbits did not have hepatitis E antibodies before infection, meeting the inclusion criteria for the experiment.

[0121] The results of HEV RNA detection in rabbit feces and HEV antigen detection in urine indicate that an animal model of hepatitis E virus infection has been successfully established. The pathological damage observed in HE staining of liver tissue after rabbit sacrifice, as well as clinical manifestations such as extrahepatic replication in various tissues, further confirm the successful establishment of the animal model.

[0122] Example 3: Evaluation of antiviral drug efficacy in the LNP-R14 rabbit model

[0123] Ribavirin treatment efficacy evaluation

[0124] Drug evaluation experimental design and flowchart as follows Figure 9As shown, rabbits in the experimental group received ribavirin via gavage the day after intravenous injection of 10 μg LNP-R14 (n = 4 / group). The gavage dose was 100 mg per rabbit per day. Fecal samples were collected weekly thereafter to detect changes in HEV RNA levels. Figure 10 .

[0125] according to Figure 10 It can be seen that HEV RNA was undetectable in the feces of rabbits in the ribavirin treatment group 21 days after LNP-R14 infection, while a large amount of HEV RNA was detected in the feces of rabbits not treated with ribavirin. This indicates that ribavirin can effectively inhibit fecal shedding caused by HEV infection in a rabbit model of HEV infection based on LNP technology, demonstrating a good antiviral effect.

[0126] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A method for constructing an animal model of hepatitis E virus infection, characterized in that, The construction method includes the following steps: A1) The recombinant vector PUC57-R14 was linearized using Hind III enzyme, purified, and then transcribed in vitro to produce viral genomic RNA; the structure of the viral genomic RNA includes a 5' non-coding region, a coding region, and a 3' non-coding region; the nucleotide sequence of the recombinant vector PUC57-R14 is SEQ ID No. 1; A2) The viral genomic RNA was mixed with the lipid excipient composition, encapsulated at room temperature, dialyzed overnight in PBS solution at 4°C, concentrated, filtered and sterilized to obtain LNP-encapsulated hepatitis E virus RNA; the lipid excipient composition includes cationic lipids, distearate phosphatidylcholine, cholesterol and PEG-lipids. A3) The hepatitis E virus RNA encapsulated in the LNP was injected intravenously into a non-human mammal; A4) Extract viral nucleic acid from fecal suspension or tissue homogenate samples of non-human mammals after inoculation and perform real-time quantitative PCR detection. When hepatitis E virus RNA is present in the sample, the hepatitis E virus infection animal model is obtained. The non-human mammal is selected from any one of rabbits, mice, and monkeys.

2. Any of the following applications of the animal model of hepatitis E virus infection constructed by the method of claim 1: B1) Application in screening anti-hepatitis E virus drugs or hepatitis E vaccines; B2) Application in evaluating the efficacy of anti-hepatitis E virus drug treatment or hepatitis E vaccine efficacy; B3) Application in functional studies of key regions or sites in the hepatitis E virus genome; B4) Application in the study of the pathogenesis of hepatitis E virus infection.

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