Construction method of animal model of hepatitis a virus infection and use thereof

Abstract

The application discloses a method for constructing an animal model infected with hepatitis A virus and application thereof. Specifically disclosed is a method for constructing an animal model infected with hepatitis A virus, which comprises the following steps: encapsulating a complete genome RNA of hepatitis A virus in LNP to obtain LNP-encapsulated hepatitis A virus genome RNA; inoculating the LNP-encapsulated hepatitis A virus genome RNA into a non-human mammal to obtain the animal model infected with the hepatitis A virus. The LNP-encapsulated HAV genome RNA is obtained through gene synthesis, and is used as inoculating material of the animal infection model, so that the LNP-encapsulated HAV genome RNA is convenient to obtain and has good uniformity and repeatability. The application first establishes an animal model infected with hepatitis A virus without isolation and culture of the virus, and the virus can be directly established and amplified, and compared with a strain, the virus genome RNA is easier to obtain and safer to operate.

Description

Technical Field

[0001] This invention belongs to the field of viral infection animal model construction technology, and relates to a method for constructing an animal model of hepatitis A virus infection and its application. Specifically, it involves using lipid nanoparticle (LNP) delivery technology to directly deliver hepatitis A virus genomic RNA into the host, successfully establishing a viral infection model. This animal model can be applied to the research and development of hepatitis A vaccines and antiviral drugs. Background Technology

[0002] Hepatitis A virus (HAV) is a single-stranded positive-sense RNA virus and one of the common pathogens causing acute hepatitis. HAV is primarily transmitted via the fecal-oral route, but can also be transmitted through contaminated water or food, or through direct contact with an infected person. The risk of infection is higher in areas lacking safe drinking water and with poor sanitation. The World Health Organization estimates that approximately 1.5 million people are infected with HAV each year; in 2016, 7,134 people died from hepatitis A globally. For a long time, human hepatitis viruses, including HAV, have struggled to replicate efficiently in cells, making it extremely difficult to obtain sufficient live virus for animal infection. Viruses obtained from fecal or serum samples of clinical patients are non-renewable, have poor homogeneity, and their virulence decreases over time, while viruses in experimentally infected animals mutate during successive passages. Therefore, the HAV research field urgently needs better infection strategies to replace live virus infection. Currently, another strategy for animal infection with HAV is the direct injection of in vitro transcribed RNA. However, since intravenous RNA degradation occurs, infection can only be achieved through intrahepatic injection. This inoculation method requires highly skilled operators, and its efficiency and uniformity are affected by its invasiveness and the easy degradation of RNA. Furthermore, as it is an invasive procedure, it poses a certain threat to animal health. Therefore, there is an urgent need for a culture-free, rapid, reliable, and minimally invasive in vivo infection method.

[0003] LNP is an efficient delivery system with great potential in mRNA vaccines, gene editing, and cancer treatment. LNP delivery systems can prevent rapid RNA degradation in vivo and significantly reduce the host's innate immune response to exogenous RNA. To date, there are no reports of establishing animal infection models by intravenously delivering viral genomic RNA into animals using LNP technology. Summary of the Invention

[0004] The technical problem to be solved by this invention is how to efficiently and rapidly construct an animal model of hepatitis A virus infection without isolating and culturing the virus. The technical problem to be solved is not limited to the technical subject matter described herein; other technical subject matter not mentioned herein will be clearly understood by those skilled in the art through the following description.

[0005] To address the aforementioned technical problems, this invention first provides a method for constructing an animal model of hepatitis A virus infection. The method includes encapsulating the complete hepatitis A virus genomic RNA in an LNP to obtain LNP-encapsulated hepatitis A virus genomic RNA, and inoculating the LNP-encapsulated hepatitis A virus genomic RNA into a non-human mammal to obtain the hepatitis A virus infection animal model.

[0006] In the above method, the structure of the complete hepatitis A virus genome RNA includes a 5' non-coding region, a coding region, and a 3' non-coding region.

[0007] In the above method, the encapsulation includes the step of mixing the genomic RNA with a lipid excipient composition, wherein the lipid excipient composition includes cationic lipids, distearate phosphatidylcholine, cholesterol, and PEG-lipids.

[0008] In the above method, the molar ratio of cationic lipid, distearate phosphatidylcholine, cholesterol and PEG-lipid in the lipid excipient composition may be, but is not limited to, 50:10:38:1.5.

[0009] In the above method, the mixing can be the mixing of the genomic RNA and the lipid excipient composition at a volume ratio of 1:2.

[0010] The cationic lipids described herein may include, but are not limited to, DLin-MC3-DMA, ALC-0315, SM-102, etc. Specifically, in one embodiment of the present invention, the cationic lipid is DLin-MC3-DMA.

[0011] The PEG-lipids mentioned in this article may include PEG. 2000 -C-DMG, ALC-0159, PEG-DMG, PEG-DSPE, PEG-DSG, PEG-dipalmitoyl, PEG-dioleyl, PEG-distearate, PEG-DAG, PEG-DPPE, PEG-PE, PEG-S-DAG, PEG-cer, etc., but not limited to these. Specifically, in one embodiment of the present invention, the PEG-lipid is PEG. 2000 -C-DMG.

[0012] The lipid excipient composition described herein may be a solution comprising cationic lipids, distearate phosphatidylcholine, cholesterol, and PEG-lipids dissolved in a solvent, wherein the solvent may be ethanol.

[0013] In the above method, the inoculation can be performed via intravenous injection.

[0014] In the above method, the non-human mammal is selected from any one of mice, rats, guinea pigs, hamsters, and monkeys.

[0015] In the above method, the non-human mammal can be a mouse.

[0016] The present invention also provides a method for constructing an animal model of hepatitis A virus infection, the method comprising the following steps:

[0017] A1) Insert the DNA fragment whose nucleotide sequence is shown in positions 1-7503 of SEQ ID No.1 in the sequence listing into the pT7 vector to obtain the recombinant vector pT7-MP4, wherein the nucleotide sequence of the recombinant vector is SEQ ID No.1;

[0018] A2) The recombinant vector pT7-MP4 was linearized using Xmal enzyme, purified by phenol-chloroform extraction, and then transcribed in vitro to produce viral genomic RNA. The viral genomic RNA comprises a 5' non-coding region (RNA transcribed from positions 1-735 of SEQ ID No. 1 in the sequence listing), a coding region (RNA transcribed from positions 736-7418 of SEQ ID No. 1 in the sequence listing), and a 3' non-coding region (RNA transcribed from positions 7419-7503 of SEQ ID No. 1 in the sequence listing).

[0019] A3) The viral genomic RNA was mixed with the lipid excipient composition, encapsulated at room temperature, dialyzed overnight in PBS solution at 4°C, concentrated and filtered to remove bacteria, and LNP-encapsulated hepatitis A virus RNA was obtained.

[0020] A4) The hepatitis A virus RNA encapsulated in the LNP was injected into mice via tail vein injection;

[0021] A5) Extract viral nucleic acid from fecal suspension or tissue homogenate samples of mice after inoculation and perform real-time quantitative PCR detection. When hepatitis A virus RNA is present in the fecal and liver tissue extracts, the animal model of hepatitis A virus infection is obtained.

[0022] In the above method, the lipid excipient composition includes cationic lipids, distearate phosphatidylcholine, cholesterol, and PEG-lipids.

[0023] This invention also provides an animal model of hepatitis A virus infection constructed by any of the methods described herein.

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

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

[0026] In the above method, the mice can be 6-8 weeks old.

[0027] This invention also provides for any of the following applications of animal models of hepatitis A virus infection constructed by any of the methods described herein:

[0028] B1) Application in screening hepatitis A vaccines or anti-hepatitis A virus drugs;

[0029] B2) Application in functional studies of key regions or sites in the hepatitis A virus genome;

[0030] Application of B3 in the study of the pathogenesis of hepatitis A virus infection.

[0031] Human hepatotropic RNA viruses (HAVs) remain difficult to culture efficiently in cell models. Strains used to establish animal infection models are generally prepared directly from clinical samples or samples collected after animal infection, resulting in inconsistent homogeneity, non-renewable specimens, and a gradual decrease in virulence. This invention discloses a method for constructing an animal model of HAV infection by delivering hepatitis A virus (HAV) genomic RNA into the host using lipid nanoparticle (LNP) delivery technology. The method utilizes the genomic DNA of the HAV strain HM175-MP4 (MP4). The MP4 genomic DNA is inserted into the pT7-HM175-18f vector, and HAV genomic RNA is obtained through in vitro transcription. This genomic RNA is then encapsulated in LNPs to synthesize LNP hepatitis A virus RNA, namely LNP-MP4, for mouse infection. After infection, viral infection characteristics are identified by monitoring fecal viral shedding, liver viral load, and liver pathological analysis. Direct intravenous injection of HAV RNA into animals fails to effectively establish infection. This invention uses LNP to encapsulate the genomic RNA of the HAV HM175-MP4 strain to prepare LNP-encapsulated hepatitis A virus RNA (LNP-HAV-mRNA), which is then delivered to C57 / B6 mice via tail vein injection. The mice exhibit typical characteristics of HAV infection, such as fecal shedding and liver inflammation, and the virus isolated from the mouse liver remains infectious.

[0032] The present invention has the following advantages: (1) It is the first time that a virus infection animal model of direct intravenous injection of HAV RNA has been established through LNP delivery technology; (2) It is the first time that an infection animal model can be directly established and the virus amplified without isolating and culturing the virus; (3) Compared with the virus strain, viral genomic RNA is easier to obtain and safer to handle; (4) LNP-encapsulated HAV genomic RNA can be obtained through gene synthesis and used as an inoculum for animal infection models. It is easy to obtain and has good uniformity and reproducibility; (5) The function of key regions or sites of the genome can be studied directly in animal models. Attached Figure Description

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

[0034] Figure 2 The results are for LNP-MP4 particle size detection in Example 1.

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

[0036] Figure 4 The changes in HAV RNA levels in the feces of mice throughout the infection process in Example 2.

[0037] Figure 5 The HAV RNA level in the liver of mice on day 42 post-infection in Example 2.

[0038] Figure 6 The results of HE staining of liver tissue from infected mice in Example 2 are shown.

[0039] Figure 7 The results of the reinfection experiment with LNP-MP4-infected mouse liver tissue extract in Example 3 are shown. Detailed Implementation

[0040] 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.

[0041] 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.

[0042] The HAV strain HM175-MP4 described in the following examples is referred to as MP4.

[0043] The pT7-HM175-18f vector in the following examples is a product of Kerafast, catalog number: ENC027.

[0044] Example 1: HAV viral genome LNP packaging

[0045] 1. Construction of an infectious cloning vector for the full-length cDNA of HAV strain MP4

[0046] The MP4 genomic DNA sequence (Genbank accession number: KX343018.1, i.e. positions 1-7503 of SEQ ID No. 1) was synthesized in vitro and double-digested with Not I and Sma I (purchased from NEB) according to the following system: 10 μg MP4 genomic DNA, 1 μl Not I, 1 μl Sma I, 10 μl 10×NEB buffer, water added to 100 μl, and reacted at 37℃ for 3 h. After enzyme digestion, the digestion products were purified using Wizard SV Gel and PCR Clean-Up System: An equal volume of membrane binding buffer was added to the reaction system, mixed well, and then added to a centrifugal adsorption column. The column was centrifuged at 13,000 rpm for 1 min. Then, 750 μl of washing buffer was added, and the column was centrifuged at 13,000 rpm for 1 min. Another 500 μl of washing buffer was added, and the column was centrifuged at 13,000 rpm for 1 min. The washing buffer was discarded, and the column was centrifuged at 13,000 rpm for 2 min. After incubation at room temperature for 1 min, 50 μl of RNase-free water was added, and the column was eluted at 13,000 rpm for 2 min to obtain the MP4 genomic DNA fragment with the enzyme digestion site.

[0047] Simultaneously, the pT7-HM175-18f vector was double-digested using the following mixture: 10 μg vector plasmid, 1 μl NotI, 1 μl SmaI, 10 μl 10×NEB buffer, and water to a final volume of 100 μl. The reaction was carried out at 37°C for 3 h. After digestion, approximately 2.8 kb of the vector fragment was recovered using agarose gel electrophoresis and named pT7. The pT7 fragment was then treated with calf intestinal alkaline phosphatase (CIP) to remove the 5′ phosphate terminus. The reaction mixture was as follows: 40 μl vector, 1 μl CIP, 5 μl 10×NEB buffer, and water to a final volume of 50 μl. The reaction was carried out at 37°C for 1 h. After purification, the vector fragment pT7 and the MP4 genomic DNA digested fragment were ligated: approximately 20 ng of vector, 200 ng of MP4 genomic DNA, 2 μl of T4 DNA ligase (200 U), 2 μl of 10× ligase buffer, and ddH2O was added to bring the volume to 20 μl. Ligation was carried out overnight at 4°C.

[0048] Transformation of Top10 competent bacteria with ligation product: Add 20 μl of ligation product to 100 μl of Top competent cells, gently stir to mix, incubate on ice for 20 min, heat shock at 42°C for 90 s, then immediately incubate on ice for 2 min, add 450 μl of antibiotic-free LB medium, and incubate at 37°C and 180 rpm for 60 min with shaking. Then, take 200 μl of bacterial culture and spread it on Amp... + Plates were incubated upside down at 37°C for 16 hours.

[0049] 2. Identification of the infectious cloning vector of the full-length cDNA of HAV strain MP4

[0050] After picking positive clones, place them in 3ml of ampoules. + The plasmid was expanded in LB medium, and plasmids were extracted using the TIAN prep MiniPlasmid Kit according to the manufacturer's instructions. The extracted plasmids were digested with Hpa I and Xho I restriction endonucleases (purchased from NEB) to verify the correct insertion sequence and orientation: 10 μl of positive plasmid, 1 μl each of Hpa I and Xho I, 2 μl of 10×NEB buffer, 0.2 μl of BSA, and water was added to a final volume of 20 μl. The mixture was incubated at 37°C for 3 h, and the digested fragments were detected by 1% agarose gel electrophoresis. The results showed that the recombinant plasmid produced fragments of 3.7 kb and 6.6 kb after double digestion with HpaI and XhoI, consistent with the expected results. The full-length cDNA cloning vector of the HAV strain MP4 was named pT7-MP4, and the nucleotide sequence of pT7-MP4 is shown in SEQ ID No. 1. Positions 1-7503 of SEQ ID No. 1 represent the nucleotide sequence of the HAV strain MP4 genomic DNA.

[0051] 3. In vitro transcription of the full-length cDNA infectious cloning vector of HAV strain MP4

[0052] pT7-MP4 was linearized using the Xmal enzyme, and the reaction system (100 μL) is shown in Table 1.

[0053] Table 1. Enzyme digestion reaction system

[0054] reagents Dosage Buffer (10×) 10ul pT7-MP4 (10ug / ul) 10ul Xmal 10ul <![CDATA[H2O]]> 70ul

[0055] The linearized plasmid was purified by phenol-chloroform extraction. The specific steps are as follows: (1) Add 200 μL of H2O to the enzyme digestion product, and then add 300 μL of DNA extraction buffer (phenol:chloroform:isoamyl alcohol = 25:24:1); (2) Centrifuge at 13000 rpm for 10 min and retain the supernatant; (3) Add an equal volume (300 μL) of nucleic acid extraction buffer (chloroform:isoamyl alcohol = 24:1), centrifuge for 5 min and retain the supernatant; (4) Add 750 μL of anhydrous ethanol, precipitate at -80℃ for 30 min, centrifuge at 13000 rpm for 10 min at 4℃ and retain the precipitate; (5) Wash once with 1 ml of 75% ethanol and centrifuge at 13000 rpm for 5 min; (6) Dry the precipitate and dissolve it in 50 μL of RNase-free H2O; (7) Measure the concentration of purified DNA using nanodrop. The purified linearized DNA (SEQ ID No. 1) was obtained.

[0056] The linearized DNA was used for in vitro transcription and capping reactions at 37°C for 6 hours. The reaction system (30 μL) is shown in Table 2 (in vitro transcription reagent was purchased from Promega, catalog number: P1300). The transcribed RNA was detected by agarose gel electrophoresis, and the results are shown below. Figure 1 The band in the second lane is the MP4 genomic RNA with a cap structure, which includes a 5' uncoding region (RNA transcribed from positions 1-735 of SEQ ID No. 1 in the sequence listing), a coding region (RNA transcribed from positions 736-7418 of SEQ ID No. 1 in the sequence listing), and a 3' uncoding region (RNA transcribed from positions 7419-7503 of SEQ ID No. 1 in the sequence listing).

[0057] Table 2. In vitro transcription reaction system

[0058] reagents Dosage 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

[0059] 4. LNP encapsulation of MP4 genomic RNA from HAV strains

[0060] 30 μg of the in vitro transcribed MP4 genomic RNA was dissolved in 20 mM citrate buffer (pH 4.0) and then mixed with a lipid excipient composition (cationic lipid (DLin-MC3-DMA), distearate phosphatidylcholine, cholesterol, and PEG-lipid (PEG)). 2000 (-C-DMG) was dissolved in ethanol at a ratio of 50:10:38:1.5 (molar ratio). The mixture was rapidly mixed at a volume ratio of 1:2 at room temperature to encapsulate the RNA in lipid nanospheres. After dialyzing overnight in PBS solution (purchased from Beijing Solarbio Science & Technology Co., Ltd., catalog number P1020) at 4°C, the mixture was concentrated, filtered, and sterilized to obtain the hepatitis A virus genomic RNA-LNP formulation. This was the synthesis of LNP-encapsulated hepatitis A virus genomic RNA, named LNP-MP4. The corresponding particle size detection results are shown in […]. Figure 2 The diameter of LNP-MP4 is approximately 100 nm.

[0061] Example 2: Verification of the infectivity of LNP-MP4 in a C57 mouse model

[0062] This embodiment utilizes the LNP-MP4 prepared in Example 1 to construct an animal model of hepatitis A virus infection, specifically LNP-MP4-infected mice, and identifies the constructed LNP-MP4-infected mice. A schematic diagram of the experimental procedure for constructing the animal model is shown below. Figure 3 As shown.

[0063] 1. Experimental design and sample collection

[0064] Five 6-8 week old C57 / B6 Ifnar1 mice were injected with LNP-MP4 as described in Example 1 via tail vein injection. - / - Mice were injected with 30 μg of LNP-MP4 and numbered 1-5 to construct LNP-MP4-infected mice.

[0065] Following viral inoculation, fecal samples were collected weekly for the detection of hepatitis A virus RNA in the feces, with continuous monitoring for 35 days. Fecal samples were prepared into a 20% (wt / vol) fecal suspension using sterile PBS (pH 7.4), centrifuged at 4°C and 12000 rpm for 5 min, and the supernatant was used for further experiments or stored at -80°C for later use. On day 42 post-inoculation, mice were euthanized and dissected. The liver was collected after dissection. Tissue samples were homogenized into a 10% (wt / vol) tissue homogenate. 0.1 g of tissue was placed in a 1.5 mL EP tube, and 1 mL of pre-chilled PBS and zirconia grinding beads were added. The homogenate was then homogenized using an automated tissue homogenizer at 4°C, 60 Hz, and 300 s. After homogenization, the homogenate was centrifuged at 12000 rpm for 5 min, and the supernatant was used for analysis. The remaining supernatant was transferred to clean EP tubes and stored at -80°C for later use. Another tissue sample was fixed in 4% paraformaldehyde fixative and sent to Seville Biotechnology Co., Ltd. for HE staining. The HE results are as follows. Figure 6 As shown.

[0066] HE staining results indicated that LNP-MP4 inoculation caused significant inflammatory cell infiltration and other damage in mouse liver tissue.

[0067] 2. HAV RNA extraction and detection

[0068] 2-1 Extraction of viral nucleic acid from liquid specimens

[0069] Viral nucleic acid was extracted from liquid specimens such as fecal suspension and tissue homogenate strictly according to the operating requirements of the nucleic acid extraction or purification kit (qEX-DNA / RNA virus, Tianlong Technology, catalog number: T387).

[0070] 2-2. Real-time quantitative PCR detection of viral RNA copy number

[0071] (1) Primers and probes

[0072] The real-time quantitative PCR (RT-qPCR) used was the TBGreen probe detection method, and the specific information of the primers is shown in Table 3.

[0073] Table 3. Primers for RT-qPCR reaction

[0074]

[0075] (2) RT-qPCR

[0076] The reaction system and parameters were prepared strictly according to the One Step TB Green PrimeScript PLUS RT-PCR Kit (TaKaRa, catalog number: RR096A) instructions. Each sample contained 20 μL of the kit, and negative and positive controls were used to ensure the reliability of the experimental results. After sample loading, the reaction tubes were placed in a Roche container. The reaction parameters were set in 480Ⅱ. After the reaction, the RNA copy number of hepatitis A virus in the sample was calculated based on the CP value of the sample calculated by the instrument and the standard curve plotted using standards. The RT-qPCR reaction system is shown in Table 4.

[0077] Table 4. RT-qPCR reaction system

[0078]

[0079]

[0080] The RT-qPCR reaction conditions were: 42℃ for 5 min, 95℃ for 10 s; 95℃ for 5 s, 60℃ for 20 s, for 40 cycles.

[0081] The results of quantitative analysis of hepatitis A virus RNA in fecal suspension are as follows: Figure 4 As shown, all five mice shed the virus in their feces within 28 days after inoculation, and mice No. 2 and No. 5 continued to shed the virus on day 35 after inoculation.

[0082] The results of hepatitis A virus RNA quantification in mouse liver tissue are as follows: Figure 5 As shown, 42 days post-infection, high viral RNA loads were detected in the livers of mice 2 and 5, consistent with viral RNA loads in feces.

[0083] Example 3: Reinfection experiment in mice using liver tissue extract from LNP-MP4-inoculated mice.

[0084] 1. Experimental design and sample collection

[0085] LNP-MP4 was injected via tail vein into three 8-week-old male C57 / B6 Ifnar1 tadpoles. - / - Mice were inoculated with a dose of 50 μg per mouse. Twenty-one days after inoculation, the mice were dissected, and liver extracts were prepared (same as in Example 2). Five C57 / B6 Ifnar1 mice were then reinfected with this extract. - / -Mice (400 μl each) were infected, and fecal samples were collected weekly to determine the viral RNA copy number, using the same method as in Example 2.

[0086] On day 14 post-infection, viral RNA was detectable in fecal extracts from three mice, such as... Figure 7 As shown in Figure A. Viral RNA was detectable in liver extracts from all mice on day 28 post-infection, as shown in Figure A. Figure 7 As shown in Figure B. The results indicate that LNP-MP4 was inoculated with C57 / B6 Ifnar1. - / - In mice, infectious hepatitis A virus particles were formed in their liver tissue. These results indicate that LNP-MP4 inoculation with C57 / B6 Ifnar1... - / - Viral RNA was continuously detected in the feces of mice. Not only was viral RNA detected in the liver tissue, but liver damage such as inflammatory cell infiltration was also observed. Furthermore, infectious hepatitis A virus particles were present in the liver tissue, indicating that we have successfully constructed an animal model of hepatitis A virus infection.

[0087] 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 A virus infection, characterized in that, The method includes the following steps: A1) The recombinant vector pT7-MP4 was linearized using Xmal enzyme, purified by phenol-chloroform extraction, and then transcribed into viral genomic RNA in vitro; 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 pT7-MP4 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 A virus RNA; the lipid excipient composition includes cationic lipids, distearate phosphatidylcholine, cholesterol and PEG-lipids. A3) The hepatitis A 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 inoculated non-human mammals and perform real-time quantitative PCR detection. When hepatitis A virus RNA is present in the sample, the hepatitis A virus infection animal model is obtained. The non-human mammals are selected from any one of mice, rats, guinea pigs, hamsters, and monkeys.

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

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