Construction method and application of hepatitis A virus infected animal model

Abstract

The invention discloses a construction method and application of a hepatitis A virus infected animal model. The invention belongs to the technical field of animal models, and particularly discloses a method for constructing a hepatitis A virus infected animal model, which comprises the following steps: encapsulating a hepatitis A virus complete genome RNA in LNP to obtain LNP encapsulated hepatitis A virus genome RNA, and inoculating the LNP encapsulated hepatitis A virus genome RNA into a non-human mammal to obtain the hepatitis A virus infected animal model. The LNP-encapsulated HAV genome RNA is obtained through gene synthesis, the LNP-encapsulated HAV genome RNA is used as an inoculation substance of an animal infection model, the LNP-encapsulated HAV genome RNA is convenient to obtain and good in uniformity and repeatability, the infected animal model can be directly established without separating and culturing the virus and the virus is amplified for the first time, and the virus genome RNA is easier to obtain and safer to operate compared with a virus strain.

Description

Technical Field

[0001] The present invention belongs to the technical field of constructing animal models of virus infection, and relates to a method for constructing an animal model of hepatitis A virus infection and uses thereof, and specifically to utilizing lipid nanoparticle (LNP) delivery technology to directly deliver hepatitis A virus genomic RNA into a host, thereby successfully establishing a virus infection model. The animal model can be applied to the research and development of hepatitis A vaccines and antiviral drugs. Background Art

[0002] Hepatitis A virus (HAV) is a single-stranded positive-strand RNA virus and one of the common pathogens that causes acute hepatitis. HAV is mainly transmitted through oral-fecal transmission, and can be transmitted through contaminated water or food, or through direct contact with infected people. The risk of infection is higher in areas with a lack of safe water and poor sanitation. According to the World Health Organization, approximately 1.5 million people are infected with HAV each year; in 2016, 7,134 people died of hepatitis A worldwide. For a long time, human hepatitis viruses, including HAV, have been difficult to replicate efficiently in cells, making it very difficult to obtain sufficient live viruses for animal infection. Viruses taken from feces and serum samples of clinical patients are non-reproducible, have poor uniformity, and their virulence decreases over time, while viruses in experimentally infected animals mutate during continuous passage. Therefore, the current HAV research field urgently needs better infection strategies to replace live virus infection. At present, another animal infection strategy for HAV is direct injection of in vitro transcribed RNA. Since intravenous RNA will degrade in large quantities, infection can only be achieved through intrahepatic injection. This inoculation method has high requirements for the operation of the experimenter. At the same time, due to its invasiveness and the easy degradation of RNA, the efficiency and uniformity of this method are affected to a certain extent. Moreover, since it is an invasive operation, it poses a certain threat to the health of animals. Therefore, there is an urgent need for an in vivo infection method that does not require culture, is fast, reliable, and has little trauma.

[0003] LNP is an effective delivery system with great potential in mRNA vaccines, gene editing and cancer treatment. The LNP delivery system can prevent the rapid degradation of RNA 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

[0004] The technical problem to be solved by the present invention is how to efficiently and quickly 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 as described, and those skilled in the art can clearly understand other technical subjects not mentioned in this article through the following description.

[0005] To solve the above technical problems, the present invention first provides a method for constructing an animal model of hepatitis A virus infection, the method comprising encapsulating the complete genomic RNA of hepatitis A virus in 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 genomic RNA of hepatitis A virus includes a 5' non-coding region, a coding region and a 3' non-coding region.

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

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

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

[0010] 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.

[0011] 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.

[0012] 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.

[0013] In the above method, the vaccination can be carried out by 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 may 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) inserting a DNA fragment having a nucleotide sequence as shown in positions 1 to 7503 of SEQ ID No. 1 in the sequence list into a pT7 vector to obtain a recombinant vector pT7-MP4, wherein the nucleotide sequence of the recombinant vector is SEQ ID No. 1;

[0018] A2) linearizing the recombinant vector pT7-MP4 using Xmal enzyme, extracting and purifying with phenol chloroform, and then transcribing viral genomic RNA in vitro; the structure of the viral genomic RNA includes a 5' non-coding region (RNA transcribed from positions 1 to 735 of SEQ ID No. 1 in the sequence list), a coding region (RNA transcribed from positions 736 to 7418 of SEQ ID No. 1 in the sequence list), and a 3' non-coding region (RNA transcribed from positions 7419 to 7503 of SEQ ID No. 1 in the sequence list);

[0019] A3) mixing the viral genomic RNA and the lipid excipient composition, encapsulating at room temperature, dialyzing in a PBS solution at 4° C. overnight, concentrating, filtering and sterilizing to obtain LNP-encapsulated hepatitis A virus RNA;

[0020] A4) inoculating mice with the LNP-encapsulated hepatitis A virus RNA by tail vein injection;

[0021] A5) extracting viral nucleic acid from the fecal suspension or tissue homogenate liquid specimen of the inoculated mice for real-time fluorescence quantitative PCR detection. When hepatitis A virus RNA is present in the feces and liver tissue extract, the hepatitis A virus infection animal model is obtained.

[0022] In the above method, the lipid excipient composition comprises cationic lipid, distearoylphosphatidylcholine, cholesterol and PEG-lipid.

[0023] The present 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 the cationic lipid, distearoylphosphatidylcholine, cholesterol and PEG-lipid in the lipid excipient composition can be 50:10:38:1.5.

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

[0026] In the above method, the mouse may be a 6-8 week old mouse.

[0027] The present invention also provides any of the following uses of the hepatitis A virus infection animal model constructed by any of the methods described herein:

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

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

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

[0031] Human hepatotropic RNA viruses are still difficult to culture efficiently in cell models. The strains used to establish animal infection models are generally prepared directly from clinical samples or samples collected after animal infection. Their uniformity is difficult to guarantee, and the specimens are non-reproducible and the virulence will gradually decrease. The present invention discloses a method for delivering hepatitis A virus (HAV) genomic RNA into a host using lipid nanoparticle (LNP) delivery technology to construct an HAV infection animal model. The genomic DNA of HAV strain HM175-MP4 (MP4 for short) is used, and the genomic DNA of MP4 is inserted into the pT7-HM175-18f vector, and the HAV genomic RNA is obtained by in vitro transcription, and the genomic RNA is encapsulated in LNP to synthesize LNP hepatitis A virus RNA, i.e., LNP-MP4, for mouse infection. After the infection is completed, the characteristics of viral infection are identified by monitoring fecal detoxification, liver viral load, and liver pathological analysis. Direct intravenous injection of HAV RNA into animals cannot effectively establish infection. The present 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 delivered to C57 / B6 mice via tail vein injection. The mice develop typical characteristics of HAV infection, such as fecal detoxification and liver inflammation, and the virus isolated from the mouse liver is still infective to the mice.

[0032] The present invention has the following advantages: (1) for the first time, a virus infection animal model of direct intravenous injection of HAV RNA is established through LNP delivery technology; (2) for the first time, an infection animal model is directly established and the virus is amplified without isolating and culturing the virus through this technology; (3) compared with the virus strain, the viral genomic RNA is easier to obtain and safer to operate; (4) LNP-encapsulated HAV genomic RNA can be obtained by gene synthesis and used as an inoculum for the animal infection model, which is easy to obtain and has good uniformity and repeatability; (5) the function of key regions or sites of the genome can be directly studied in the animal model. 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 LNP-MP4 in Example 1.

[0035] Figure 3 Schematic diagram of the overall experimental process in Example 2.

[0036] Figure 4 The figure shows the changes in HAV RNA levels in mouse feces during the entire infection process in Example 2.

[0037] Figure 5 This is the HAV RNA level in the liver of mice on the 42nd day after infection in Example 2.

[0038] Figure 6 This is the HE staining result of liver tissue of mice in the infection group in Example 2.

[0039] Figure 7 These are the results of the reinfection experiment with the liver tissue extract of mice infected with LNP-MP4 in Example 3. DETAILED DESCRIPTION

[0040] 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.

[0041] 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.

[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 virus genome LNP packaging

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

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

[0047] At the same time, the pT7-HM175-18f vector was double-digested according to the following system: 10 μg of vector plasmid, 1 μl of NotI, 1 μl of SmaI, 10 μl of 10×NEB buffer, water to 100 μl, and react at 37°C for 3 hours. After digestion, the vector fragment of about 2.8 kb was recovered using agarose gel, and the vector fragment was named pT7. The vector fragment pT7 was treated with calf intestinal alkaline phosphatase (CIP) to remove the 5′ phosphate end of the vector fragment. The system is as follows: 40 μl of vector, 1 μl of CIP, 5 μl of 10×NEBufer 3, water to 50 μl system, and react at 37°C for 1 hour. After purification, the vector fragment pT7 and the MP4 genomic DNA fragment were ligated: about 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 the volume was made up to 20 μl with ddH2O, and the ligation was carried out at 4°C overnight.

[0048] Transformation of ligation product into Top10 competent bacteria: Add 20μl ligation product to 100μl Top competent cells, gently stir to mix, ice bath for 20min, heat shock at 42℃ for 90s, quickly ice bath for 2min, add 450μl LB medium without antibiotics, shake and culture at 37℃ and 180rpm for 60min. Then take 200μl of bacterial solution and apply Amp + The plate was inverted and incubated at 37°C for 16 h.

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

[0050] After picking the positive clones, place them in 3 ml Amp + The culture was expanded in LB medium, and the plasmid was extracted using TIAN prep MiniPlasmid Kit according to the instructions. The extracted plasmid was digested and identified using Hpa I and Xho I restriction endonucleases (purchased from NEB) to verify whether the insertion sequence and direction were correct: 10 μl of positive plasmid, 1 μl of Hpa I and Xho I, 2 μl of 10×NEB buffer, 0.2 μl of BSA, and water was added to 20 μl. The reaction was carried out in a 37°C water bath for 3 hours, and the digested fragments were detected by 1% agarose gel electrophoresis. The results showed that the recombinant plasmid was double-digested by HpaI and XhoI to produce 3.7kb and 6.6kb fragments, which were consistent with the expected results. The full-length cDNA cloning vector of the HAV strain MP4 genome was named pT7-MP4, and the nucleotide sequence of pT7-MP4 is shown in SEQ ID No.1. Among them, the 1st to 7503rd positions of SEQ ID No.1 are the nucleotide sequence of the genomic DNA of the HAV strain MP4.

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

[0052] pT7-MP4 was linearized using Xmal enzyme, and the reaction system (100ul) 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 200ul of H2O to the digested product, and then add 300ul of DNA extract (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 extract (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, add 50ul of RNase free H2O to dissolve; (7) Use nanodrop to measure the concentration of purified DNA. The linearized DNA (SEQ ID No.1) was purified.

[0056] The above linearized DNA was used for in vitro transcription and capping reaction 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 was detected by agarose electrophoresis. The results are shown in Table 2. Figure 1 The band in the second lane is the MP4 genomic RNA with a cap structure, and its structure includes a 5' non-coding region (RNA transcribed from positions 1-735 of SEQ ID No. 1 in the sequence list), a coding region (RNA transcribed from positions 736-7418 of SEQ ID No. 1 in the sequence list), and a 3' non-coding region (RNA transcribed from positions 7419-7503 of SEQ ID No. 1 in the sequence list).

[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 HAV Strain MP4 Genomic RNA

[0060] 30 μg of the in vitro transcribed MP4 genomic RNA 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 quickly mixed at a volume ratio of 1:2 at room temperature to encapsulate RNA in the lipid nanosphere preparation; after overnight dialysis at 4°C in PBS solution (purchased from Beijing Solebow Technology Co., Ltd., catalog number P1020), the hepatitis A virus genomic RNA-LNP preparation was obtained after concentration, filtration and sterilization, that is, the hepatitis A virus genomic RNA encapsulated by LNP was synthesized and named LNP-MP4. The corresponding particle size test results are shown in Figure 2 , the diameter of LNP-MP4 is about 100 nm.

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

[0062] In this example, the LNP-MP4 prepared in Example 1 was used to construct an animal model of hepatitis A virus infection, i.e., LNP-MP4 infected mice, and the constructed LNP-MP4 infected mice were identified. The experimental flow diagram of the animal model construction is shown in FIG. Figure 3 shown.

[0063] 1. Experimental design and sample collection

[0064] The LNP-MP4 in Example 1 was injected into the tail vein of 5 6-8 week old C57 / B6 Ifnar1 - / - Mice were injected with 30ug of LNP-MP4 per mouse, which were numbered 1-5 to construct LNP-MP4-infected mice.

[0065] After virus inoculation, fecal specimens were collected once a week for detection of hepatitis A virus RNA in feces, and continuous monitoring was performed for 35 days. The fecal specimens were prepared into a 20% (wt / vol) fecal suspension with sterile PBS (pH 7.4), centrifuged at 4°C and 12000rpm for 5min, and the supernatant was taken for the next experiment or stored in a -80°C refrigerator for later use. On the 42nd day after virus inoculation, the mice were euthanized and dissected. The liver was collected after dissection. 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, and the tissue was ground with an automatic tissue grinder, with the parameters set to 4°C, 60Hz, and 300s. After grinding, centrifuged at 12000rpm for 5min, the supernatant was taken for detection, and the remaining supernatant was transferred to a clean EP tube and frozen in a -80°C refrigerator for later use. Another tissue sample was fixed in 4% paraformaldehyde and sent to Sewell Biotechnology Co., Ltd. for HE staining. The HE results are as follows: Figure 6 shown.

[0066] HE staining results indicated that LNP-MP4 inoculation could cause obvious inflammatory cell infiltration and other damages 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 acids were extracted from fecal suspensions, tissue homogenates and other liquid specimens strictly according to the operating requirements of the nucleic acid extraction or purification kit (qEX-DNA / RNA Virus, Tianlong Technology, Catalog No.: T387).

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

[0071] (1) Primers and probes

[0072] The reverse transcription 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. RT-qPCR reaction primers

[0074]

[0075] (2) RT-qPCR

[0076] Strictly follow the instructions of One Step TB Green PrimeScript PLUS RT-PCR Kit (TaKaRa, Catalog No.: RR096A) to prepare the system and set the reaction parameters. A total of 20 μL of the system was used for each sample, and negative and positive controls were used to ensure the reliability of the experimental results. After the sample was loaded, the reaction tube was placed in the Roche The reaction parameters were set in 480Ⅱ. After the reaction was completed, 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 drawn using the standard. 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°C for 5 min, 95°C for 10 s; 95°C for 5 s, 60°C for 20 s, for 40 cycles.

[0081] Quantitative results of HAV RNA in fecal suspensions are as follows Figure 4 As shown, within 28 days after inoculation, all five mice excreted the virus in their feces, and mice No. 2 and No. 5 continued to excrete the virus on the 35th day after inoculation.

[0082] The quantitative results of HAV RNA in mouse liver tissue are shown in Figure 5 As shown, 42 days after infection, higher viral RNA loads were detected in the livers of mice No. 2 and No. 5, which was consistent with the viral RNA loads in feces.

[0083] Example 3: Mouse reinfection experiment with liver tissue extracts of mice inoculated with LNP-MP4

[0084] 1. Experimental design and sample collection

[0085] Three 8-week-old male C57 / B6 Ifnar1 mice were inoculated with LNP-MP4 via tail vein injection. - / - Mice were inoculated with 50 μg of the vaccine per mouse. On day 21 of the inoculation, the mice were dissected and liver extracts were prepared (same as in Example 2). The extracts were used to infect 5 C57 / B6 Ifnar1 mice again. - / -Mice (each infected with 400 μl) were inoculated, and mouse feces were collected weekly to determine the number of viral RNA copies, using the same detection method as in Example 2.

[0086] On the 14th day after reinfection, viral RNA was detected in the fecal extracts of three mice, e.g. Figure 7 As shown in A. On the 28th day after reinfection, viral RNA was detected in the liver extracts of all mice, as shown in Figure 7 As shown in B. The results show that LNP-MP4 inoculated C57 / B6 Ifnar1 - / - The above results show that LNP-MP4 vaccination of C57 / B6 Ifnar1 - / - Viral RNA can be continuously detected in the feces of mice. Not only can viral RNA be detected in the liver tissue, but liver damage such as inflammatory cell infiltration is also present. In addition, infectious hepatitis A virus particles are 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. 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 A virus infection, characterized in that: The method comprises encapsulating the complete genomic RNA of hepatitis A virus in LNP to obtain the 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.

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

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

4. The method according to any one of claims 1 to 3, characterized in that: The non-human mammal is selected from any one of mice, rats, guinea pigs, hamsters and monkeys.

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

6. A method for constructing an animal model of hepatitis A virus infection, characterized in that: The method comprises the following steps: A1) inserting a DNA fragment having a nucleotide sequence as shown in positions 1 to 7503 of SEQ ID No. 1 in the sequence list into a pT7 vector to obtain a recombinant vector pT7-MP4, wherein the nucleotide sequence of the recombinant vector is SEQ ID No. 1; A2) linearizing the recombinant vector pT7-MP4 using Xmal enzyme, extracting and purifying with phenol chloroform, and then transcribing the 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; A3) mixing the viral genomic RNA and the lipid excipient composition, encapsulating at room temperature, dialyzing in a PBS solution at 4° C. overnight, concentrating, filtering and sterilizing to obtain LNP-encapsulated hepatitis A virus RNA; A4) inoculating mice with the LNP-encapsulated hepatitis A virus RNA by tail vein injection; A5) extracting viral nucleic acid from the fecal suspension or tissue homogenate liquid specimen of the inoculated mice for real-time fluorescence quantitative PCR detection. When hepatitis A virus RNA is present in the feces and liver tissue extract, the hepatitis A virus infection animal model is obtained.

7. The method according to claim 6, characterized in that The lipid excipient composition includes a cationic lipid, distearoylphosphatidylcholine, cholesterol and PEG-lipid.

8. An animal model of hepatitis A virus infection constructed by the method described in any one of claims 1-5 or 6-7.

9. Any of the following uses of the hepatitis A virus infection animal model constructed by the method of any one of claims 1 to 5 or 6 to 7: B1) Application in screening hepatitis A vaccine or anti-hepatitis A virus drugs; B2) Application in functional studies of key regions or sites of the hepatitis A virus genome; B3) Application in the study of the pathogenesis of hepatitis A virus infection.

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