Construction method and application of hepatitis E virus infected animal model

Abstract

The invention discloses a construction method and application of a hepatitis E virus infected animal model. The invention belongs to the technical field of animal models, and particularly discloses a construction method of a hepatitis E virus infected animal model, which comprises the following steps: encapsulating a complete hepatitis E virus genome RNA in LNP to obtain an LNP-encapsulated hepatitis E virus genome RNA, and inoculating the LNP-encapsulated hepatitis E virus genome RNA into a non-human mammal to obtain the hepatitis E virus infected animal model. The animal infection model can be established when the RNA dosage is as low as 1ug. The technical barrier that the hepatitis E virus is not suitable for in-vitro culture is solved, the virus does not need to be separated from natural or clinical specimens, the LNP-encapsulated HEV genome RNA can be obtained through gene synthesis, and the LNP-encapsulated HEV genome RNA serving as an inoculation substance of an animal infection model is convenient to obtain, good in uniformity and repeatability and safer to operate.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a method for constructing an animal model infected with hepatitis E virus and an application thereof. Background Art

[0002] Hepatitis E virus (HEV) belongs to the Hepatitis Eviridae family. It is a single-stranded positive-strand RNA virus that is transmitted through the fecal-oral route and is one of the main pathogens causing acute and chronic hepatitis. In areas with high HEV prevalence, the total incidence of hepatitis E is 1%-15%. Most clinical symptoms of hepatitis E are self-limiting acute infections, but pregnant women infected with HEV have a high rate of fulminant liver disease (acute liver failure) and a mortality rate of nearly 30%. In addition, HEV can cause persistent infection in organ transplant patients and immunocompromised patients, and this chronic hepatitis caused by HEV has become an important clinical problem. Due to the current lack of efficient animal infection models and in vitro cell culture systems, research on HEV pathogenic mechanisms, vaccine and antiviral drug development has been greatly limited.

[0003] HEV is extremely difficult to culture in vitro. Although a few studies have reported that wild HEV strains can be cultured in vitro, they are difficult to replicate. Therefore, the source of inoculum for the construction of the commonly used HEV infection animal model is mostly the fecal suspension or serum of 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 the animal body, there is still the problem of viral genome mutation, which causes the virulence to decrease over time, and the uniformity cannot be guaranteed. Therefore, obtaining a stable virus seed liquid is the bottleneck for the construction of HEV infection animal models. In recent years, it has been reported that the in vivo infection characteristics of HEV can be replicated by intrahepatic injection of in vitro transcribed RNA of HEV. However, intrahepatic injection is traumatic, and the infection efficiency of this method is low. Therefore, it is urgent to develop a rapid, cell culture-free, and less traumatic HEV infection animal model.

[0004] Lipid nanoparticles (LNP) are an effective delivery system with great potential in mRNA vaccines, gene editing and cancer treatment. LNP delivery systems can prevent rapid RNA degradation in the body and greatly reduce the host's natural immune response to exogenous RNA. So far, there has been no report on the intravenous delivery of viral genomic RNA into animals based on LNP technology to establish an infected animal model. Summary of the invention

[0005] The technical problem to be solved by the present invention is how to construct an animal model of hepatitis E virus infection. The technical problem to be solved is not limited to the technical subject as described, and those skilled in the art can clearly understand other technical subjects not mentioned in this article through the following description.

[0006] To solve the above technical problems, the present invention first provides a method for constructing an animal model of hepatitis E virus infection. The construction method may include encapsulating the complete genomic RNA of hepatitis E virus in LNP to obtain LNP-encapsulated hepatitis E virus genomic RNA, and inoculating the LNP-encapsulated hepatitis E virus genomic RNA into a non-human mammal to obtain the hepatitis E virus infection animal model.

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

[0008] In the above construction method, the structure of the complete genomic RNA of hepatitis E virus may 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 rabbit, mouse and monkey.

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

[0011] Furthermore, the encapsulation comprises the step of mixing the complete genomic RNA of hepatitis E virus with a lipid excipient composition, wherein the lipid excipient composition comprises a cationic lipid, distearoylphosphatidylcholine, cholesterol and PEG-lipid.

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

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

[0014] The cationic lipids described herein may include DLin-MC3-DMA, ALC-0315, SM-102, etc. but are not limited thereto. Specifically, in one embodiment of the present invention, the cationic lipid is DLin-MC3-DMA.

[0015] The PEG-lipids described herein may include PEG 2000-C-DMG, ALC-0159, PEG-DMG, PEG-DSPE, PEG-DSG, PEG-dipalmitoleyl, PEG-dioleyl, PEG-distearyl, PEG-DAG, PEG-DPPE, PEG-PE, PEG-S-DAG, PEG-cer, etc., but not limited thereto. Specifically, in one embodiment of the present invention, the PEG-lipid is PEG 2000 -C-DMG.

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

[0017] The present invention also provides a method for constructing an animal model of hepatitis E virus infection, and the method may include the following steps:

[0018] A1) linearizing the recombinant vector PUC57-R14 using Hind III enzyme, and transcribing the viral genomic RNA in vitro after purification; 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;

[0019] A2) mixing the viral genomic RNA with a lipid excipient composition, encapsulating at room temperature, dialyzing in a PBS solution at 4° C. overnight, and then concentrating, filtering and sterilizing to obtain LNP-encapsulated hepatitis E virus RNA; the lipid excipient composition comprises a cationic lipid, distearoyl phosphatidylcholine, 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 specimen of the inoculated non-human mammal for real-time fluorescence quantitative PCR detection; when hepatitis E virus RNA is present in the specimen, the hepatitis E virus-infected animal model is obtained.

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

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

[0024] In the above construction method, the molar ratio of the cationic lipid, distearoyl phosphatidylcholine, cholesterol and PEG-lipid in the lipid excipient 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 2000 -C-DMG.

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

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

[0028] B2) Application in the evaluation of the therapeutic effects of anti-hepatitis E virus drugs or hepatitis E vaccine;

[0029] B3) Application in functional studies of key regions or sites of the hepatitis E virus genome;

[0030] B4) Application in the study of the pathogenesis of hepatitis E virus infection.

[0031] The present invention also provides an animal model of hepatitis E virus infection, and the animal model of hepatitis E virus infection can be constructed by any construction method described herein.

[0032] The present invention discloses a synthetic LNP hepatitis E virus RNA (LNP-R14) obtained by encapsulating the hepatitis E virus RNA genome with lipid nanoparticles (LNP), and a method for directly infecting animals with the synthetic LNP hepatitis E virus RNA, which can make animals show typical virus infection characteristics, and the animal infection model can be established with an RNA dosage as low as 1ug. After the infection is completed, the virus infection characteristics are identified by monitoring fecal detoxification, urine antigens, liver viral load, and liver pathological analysis. The animal model can be applied to the evaluation of anti-HEV drugs and vaccines. The present invention also discloses a use of the above-mentioned animal infection model for in vivo evaluation of hepatitis E virus vaccines and antiviral drugs. The present invention has the following advantages: (1) It solves the technical barrier that hepatitis E virus is not suitable for in vitro culture; (2) There is no need to isolate the virus from nature or clinical specimens, and the LNP-encapsulated HEV genome RNA can be obtained by gene synthesis as the inoculation material of the animal infection model, which is convenient to obtain and has good uniformity and repeatability. Compared with viral strains, viral genomic RNA is safer to manipulate; (3) synthetic LNP hepatitis E virus RNA can be directly genetically modified to study the functions of key regions or sites of the viral genome in animal infection models. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The results of the in vitro transcription experiment in Example 1 are shown.

[0034] Figure 2 This is the particle size detection result of R14 LNP in Example 1.

[0035] Figure 3 Schematic diagram of the overall experimental process of 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 the change of HEV RNA level in the feces of each group of rabbits during the whole infection process in Example 2.

[0038] Figure 6 This is the change of HEV ORF2 antigen level in the urine of each group of rabbits during the whole infection process in Example 2.

[0039] Figure 7 HEV in different tissues and organs of rabbits in each group on the 21st day after infection in Example 2 RNA levels.

[0040] Figure 8 These are the HE staining results of liver tissues of rabbits in each group in Example 2.

[0041] Fig. 9 This is the design and flow chart of the antiviral drug effect evaluation in Example 3.

[0042] Fig.10 To evaluate the effect of antiviral drugs in Example 3, the levels of HEV RNA in rabbit feces were changed. DETAILED DESCRIPTION

[0043] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0044] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0045] Example 1: HEV viral genome RNA LNP encapsulation

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

[0047] The R14 infectious cloning vector was linearized using Hind III enzyme, and the reaction system (100ul) is shown in Table 1. The sequence of the R14 plasmid (R14 infectious cloning vector PUC57-R14) is shown in SEQ ID No.1.

[0048] Table 1. R14 plasmid linearization reaction system

[0049] Reagents Dosage Buffer(10x) 10ul PUC57-R14 100ug / 10ul Hind III 10ul <![CDATA[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 digested 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℃ and 13000rpm for 10min, and retain the precipitate; (5) Add 1ml of 75% ethanol to wash once, and centrifuge at 13000rpm for 5min; (6) Dry the precipitate and add 50ul of RNase free H2O to dissolve it; (7) Use nanodrop to measure the concentration of purified DNA.

[0051] The linearized DNA was used for in vitro transcription at 37°C for 6 hours. The reaction system (30ul system) is shown in Table 2 (in vitro transcription reagent purchased from Promega, catalog number: P1300). The in vitro transcribed RNA can be detected by agarose electrophoresis. The results are shown in Table 2. Figure 1 , R14 has two bands from top to bottom, the first one is the DNA template and the second one is the in vitro transcribed RNA.

[0052] Table 2. In vitro transcription reaction system

[0053] 5×Buffer 6ul rNTP 6ul (A:C:U:G=3:3:3:1 mixture) Cap 2ul T7 enzymix 2.5ul Linearized DNA 1ug <![CDATA[ddH2O]]> 14.5ul

[0054] 2. HEV strain R14 genome RNA LNP encapsulation

[0055] 30 μg of the R14 genomic RNA obtained by in vitro transcription was dissolved in 20 mM citric acid buffer (pH = 4.0) and then mixed with a lipid excipient composition (cationic lipid (DLin-MC3-DMA), distearoyl phosphatidylcholine, cholesterol and PEG-lipid (PEG 2000-C-DMG) was dissolved in ethanol at a ratio of 50:10:38:1.5 (molar ratio) and mixed quickly at a volume ratio of 1:2 at room temperature to encapsulate the R14 genomic RNA in the lipid nanosphere preparation; after overnight dialysis at 4°C in a PBS solution (purchased from Beijing Solebow Technology Co., Ltd., item number P1020), the hepatitis E virus genomic RNA-LNP preparation was obtained after concentration, filtration and sterilization, that is, 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 is 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, an animal model of hepatitis E virus infection, i.e., LNP-R14 infected rabbit (also referred to as LNP-R14 rabbit model), was constructed, and the constructed LNP-R14 infected rabbit was identified. The overall experimental flow diagram of the animal model construction is shown in FIG. Figure 3 shown.

[0058] 1. Animal enrollment and screening

[0059] Japanese white rabbits (2.5-3.5 months old, half male and half female, weighing about 2.5-3 kg) were purchased from Beijing Jinmuyang Experimental Breeding Co., Ltd. All rabbits were housed in separate cages and given adequate water and feed. The animal ethics review of the experiment was passed by the Animal Ethics Committee of Peking University Health Science Center before the experiment.

[0060] Before the experiment, serum and fecal specimens were collected from all experimental rabbits. HEV RNA in feces was detected by RT-qPCR, and anti-HEV antibodies in serum were detected by enzyme-linked immunosorbent assay (ELISA). Rabbits with negative anti-HEV antibodies and HEV RNA were selected for inclusion in the group.

[0061] 2. Experimental design and sample collection

[0062] The HEV RNA (LNP-R14) coated with nanoparticles was inoculated into large-eared white rabbits by intravenous injection. The injection method was as shown in Table 3 and Figure 4 shown.

[0063] Table 3. Experimental groups and inoculation doses

[0064]

[0065]

[0066] After virus inoculation, urine, blood and feces samples were collected once a week for the detection of virological indicators such as fecal HEV RNA, urine HEV antigen and serum anti-HEV antibody, and continuous monitoring. The fecal samples were prepared into a 20% (wt / vol) fecal suspension with sterile PBS (pH7.4), centrifuged at 4°C and 5000rpm for 20min, and the supernatant was taken for the next experiment or stored in a -80°C refrigerator for later use. The blood samples were centrifuged at 4°C and 5000rpm for 20min to obtain serum for the next experiment or stored in a -80°C refrigerator for later use. At the third week after virus inoculation (21dpi), 4 rabbits were selected from each group for euthanasia and dissection. After dissection, the liver, kidney, duodenum, urine, and bile of the rabbits were collected, and one rabbit from each group was selected for heart and brain. Tissue samples were made into 10% (wt / vol) tissue homogenate. First, 0.1g of tissue was taken and placed in a 1.5mL EP tube. 1mL of pre-cooled PBS and zirconium oxide grinding beads were added. The mixture was ground with an automatic tissue grinder. The parameters were set to 4°C, 60Hz, and 60s. This was repeated 5 times, with a 9s interval between each time. After grinding, the mixture was centrifuged at 8000rpm for 5min. The supernatant was taken for detection. The remaining supernatant was transferred to a clean EP tube and stored in a -80°C refrigerator for later use. Another portion of the tissue sample was fixed in 10% formalin fixative for 24h and sent to Seville Biotechnology Co., Ltd. for HE staining experiments. The HE results are as follows: Figure 8 shown.

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

[0068] 3. HEV RNA extraction and detection

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

[0070] Strictly follow The operation of Viral DNA / RNA Kit (Quanshijin) requires the extraction of viral nucleic acid from liquid specimens such as fecal suspension, tissue homogenate and bile. The detailed steps are as follows:

[0071] 1) Add 20 μL Proteinase K and 200 μL BB5 to a sterile 1.5 mL EP tube that has been treated without RNAse. Vortex and mix for 15 seconds (for multiple samples, Proteinase K and BB5 can be mixed in a ratio of 1:10 and aliquoted into 220 μL for use).

[0072] 2) Add 200 μL of sample into the EP tube, vortex and mix for 15 seconds, and place in a 56°C water bath for 15 minutes.

[0073] 3) Take out 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 leave it at room temperature for 5 minutes.

[0074] 4) Add the solution to a centrifuge column (the centrifuge column is placed in a collection tube), centrifuge at room temperature and 12,000 g for 1 min, and discard the effluent in the collection tube.

[0075] 5) Add 500 μL WB5 (make sure anhydrous ethanol has been added before use) to the centrifuge column, centrifuge at room temperature and 12,000 g 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 the remaining WB5 to prevent the remaining WB5 from inhibiting subsequent reactions.

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

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

[0080] 10) The RNA was immediately subjected to reverse transcription reaction or stored in a -80°C refrigerator for later use.

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

[0082] (1) Primers and probes

[0083] Reverse transcription real-time quantitative PCR (RT-qPCR) is a Taqman probe detection method, wherein the 3' end of the probe is labeled with BHQ-1 and the 5' end is labeled with 6-FAM. The specific information of 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 follow Probe 1-Step RT-qPCR System Kit (Promega, Catalog No. A6121) was used to prepare the system and set the reaction parameters. Each sample had a total of 20 μL of the system, and each sample was set up with 2 independent replicate wells. After the sample was loaded, the wells were placed in the Roche 480Ⅱ and set the reaction parameters; after the reaction, use negative and positive controls to ensure the reliability of the experimental results. According to the CP values ​​of the samples and standard plasmids calculated by the instrument, the two standard plasmids with known copy numbers correspond to the copy numbers of the standard curve in sequence. The instrument will automatically calculate the copy number of the sample according to the CP value of the sample, record the copy number of the two replicate wells of the sample, and convert it to the HEV copy number of the corresponding sample after calculating the average value. The reaction system of RT-qPCR is shown in Table 5.

[0088] Table 5. RT-qPCR reaction system

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

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

[0091] Fecal suspension quantitative results such as Figure 5 As shown, within three weeks of inoculation with LNP-R14, all eight rabbits in the 100μg and 10μg groups showed fecal excretion, only one rabbit in the 1μg group did not excrete, and no fecal excretion was detected in the 0.1μg group, proving that LNP-R14 can effectively establish infection and cause fecal excretion in the rabbit model.

[0092] Rabbit tissue quantitative results are as follows Figure 7 As shown, the liver, kidney, small intestine, heart and bile of all rabbits sacrificed in the 100 μg group were positive for HEV RNA, and some rabbit tissues in the 10 μg and 1 μg groups were positive for nucleic acid, indicating that HEV replication can occur in a variety of extrahepatic tissues of rabbits after inoculation with LNP-R14.

[0093] 4. Detection of HEV antigen

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

[0095] 1) Preparation: Dilute 50 mL of 20× concentrated detergent to 1 L with deionized water.

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

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

[0098] 4) Incubation: Seal the plate with a sealing film and place in a 37°C incubator for 1 hour.

[0099] 5) Washing: Wash the plate 5 times using a plate washer and dry it after washing.

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

[0101] 7) Incubation: Seal the plate with a sealing film and place in a 37°C incubator for 30 min.

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

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

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

[0105] 11) Result determination: Critical value = 0.12 + average value of negative control well A. If the sample A value > critical value, it is determined to be positive; if the sample A value ≤ critical value, it is determined to be negative. S / CO (signal to cutoff) value calculation: sample A value / critical value. Similarly, when the S / CO value > 1, it is determined to be positive, and when the S / CO value ≤ 1, it is determined to be negative.

[0106] Antigen test results Figure 6 As shown, on the 21st day after infection, all eight rabbits in the 100μg group were positive for urine antigen, some rabbits in the 10μg and 1μg groups were positive for urine antigen, and all rabbits in the 0.1μg group were negative for urine antigen. The results showed that LNP-R14 infection in rabbits can result in a dose-related positive urine antigen phenomenon.

[0107] 5. Detection of anti-HEV antibodies

[0108] The serum samples were all tested for anti-HEV total antibodies using the ELISA double antigen sandwich method. The experimental process was strictly carried out according to the instructions of the Hepatitis E Virus Antibody Detection Kit (Beijing Wantai, product batch number: ES20230201A). The specific steps are as follows:

[0109] 1) Preparation: Dilute 50 mL of 20× concentrated detergent to 1 L with deionized water.

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

[0111] 3) Dilution and sample addition: First add 50 μL of sample diluent to each well, then add 50 μL of the sample to be tested, negative or positive control.

[0112] 4) Incubation: Seal the plate with a sealing film and place in a 37°C incubator for 30 min.

[0113] 5) Washing: Wash the plate 5 times using a plate washer and dry it after washing.

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

[0115] 7) Incubation: Seal the plate with a sealing film and place in a 37°C incubator for 30 min.

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

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

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

[0119] 11) Result determination: critical value = 0.12 + negative control A mean. If the sample A value > critical value, it is determined to be positive; if the sample A value ≤ critical value, it is determined to be negative. S / CO value calculation: sample A value / critical value. Similarly, when the S / CO value > 1, it is determined to be positive, and when the S / CO value ≤ 1, it is determined to be negative.

[0120] The antibody test results proved that the rabbits had no hepatitis E antibodies before infection and met the experimental inclusion criteria.

[0121] The results of HEV RNA detection in rabbit feces and HEV antigen detection in urine indicate that the animal model of hepatitis E virus infection has been successfully established. The clinical manifestations of pathological damage in liver tissue HE staining and extrahepatic replication of various tissues after the rabbits were killed also prove the successful establishment of the animal model.

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

[0123] Evaluation of the therapeutic effect of ribavirin

[0124] Drug evaluation experimental design and flow chart Fig. 9As shown in Figure 2, rabbits in the experimental group were intravenously injected with LNP-R14 10 μg (n=4 / group) and then given ribavirin by gavage the next day. The gavage dose was 100 mg per rabbit per day. Fecal samples were collected weekly and the changes in HEV RNA levels were detected. Fig.10 .

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

[0126] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A method for constructing an animal model of hepatitis E virus infection, characterized in that: The construction method comprises encapsulating the complete genomic RNA of hepatitis E virus in LNP to obtain the LNP-encapsulated hepatitis E virus genomic RNA, and inoculating the LNP-encapsulated hepatitis E virus genomic RNA into a non-human mammal to obtain the hepatitis E virus infection animal model.

2. The construction method according to claim 1, characterized in that: The vaccination method is intravenous injection.

3. The construction method according to claim 1 or 2, characterized in that: The structure of the complete genome RNA of hepatitis E virus includes a 5' non-coding region, a coding region and a 3' non-coding region.

4. The construction method according to any one of claims 1 to 3, characterized in that: The non-human mammal is selected from any one of rabbit, mouse and monkey.

5. The construction method according to any one of claims 1 to 4, characterized in that: The non-human mammal is a rabbit.

6. A method for constructing an animal model of hepatitis E virus infection, characterized in that: The construction method comprises the following steps: A1) linearizing the recombinant vector PUC57-R14 using Hind III enzyme, and transcribing the viral genomic RNA in vitro after purification; 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) mixing the viral genomic RNA with a lipid excipient composition, encapsulating at room temperature, dialyzing in a PBS solution at 4° C. overnight, and then concentrating, filtering and sterilizing to obtain LNP-encapsulated hepatitis E virus RNA; the lipid excipient composition comprises a cationic lipid, distearoyl phosphatidylcholine, cholesterol and PEG-lipid; A3) inoculating a non-human mammal with the LNP-encapsulated hepatitis E virus RNA by intravenous injection; A4) extracting viral nucleic acid from a fecal suspension or tissue homogenate liquid specimen of the inoculated non-human mammal for real-time fluorescence quantitative PCR detection; when hepatitis E virus RNA is present in the specimen, the hepatitis E virus-infected animal model is obtained.

7. The construction method according to claim 6, characterized in that: The non-human mammal is selected from any one of rabbit, mouse and monkey.

8. The construction method according to claim 7, characterized in that: The non-human mammal is a rabbit.

9. Any of the following uses of the hepatitis E virus infection animal model constructed by the construction method of any one of claims 1 to 5 or 6 to 8: B1) Application in screening of anti-hepatitis E virus drugs or hepatitis E vaccines; B2) Application in the evaluation of the therapeutic effects of anti-hepatitis E virus drugs or hepatitis E vaccine; B3) Application in functional studies of key regions or sites of the hepatitis E virus genome; B4) Application in the study of the pathogenesis of hepatitis E virus infection.

10. A hepatitis E virus infection animal model, characterized in that: The hepatitis E virus infection animal model is constructed by the construction method described in any one of claims 1-5 or 6-8.

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