Construction method and application of mouse model with hemorrhagic fever with renal syndrome

Abstract

The invention belongs to the technical field of construction methods of disease animal models, and particularly relates to a construction method and application of a renal syndrome hemorrhagic fever mouse model. According to the HTNV infected mouse model established by the method disclosed by the invention, the symptom of the experimental mouse is close to the clinical symptom of a severe hemorrhagic fever with renal syndrome after challenge, the complete lethality and repeatability are high, and the HTNV infected mouse model has the advantages of simple operation, low cost and high efficiency. The method can be used for research on the HTNV pathogenic mechanism and immune mechanism and research and development of antiviral drugs and vaccines, and has great application value for perfecting an HTNV full-chain prevention and control system.

Description

Technical Field

[0001] This invention relates to the technical field of methods for constructing animal models of diseases, specifically to a method for constructing and applying a mouse model of hemorrhagic fever with renal syndrome. Background Technology

[0002] Hantaan virus (HTNV) belongs to the order Elliotviruses (Erliovirales) Elliovirale ), Hantaviridae ( Hantaviridae ), Orthohantavirus genus ( Orthohantavirus Members of the family of hemorrhagic fever with renal syndrome (HFRS), primarily cause this condition. HFRS is clinically characterized by fever, hemorrhage, and acute kidney injury. It is widespread, has a high mortality rate, and poses a severe threat. Currently, there are no specific antiviral drugs for HFRS, and existing inactivated vaccines suffer from poor duration of immune protection, insufficient cross-protection between subtypes, and poor adherence to repeated immunizations. Therefore, a deeper understanding of the pathogenesis of HTNV and the development of effective antiviral drugs and novel vaccines is urgently needed. Exploring the pathogenesis and interventions of HTNV requires the use of platforms such as in vitro experiments, animal experiments, and clinical trials. Animal experiments serve as a bridge between in vitro and clinical trials, verifying in vitro findings and providing fundamental data for clinical trials. Therefore, establishing animal models that can mimic the characteristics of human HFRS is fundamental to studying viral pathogenesis and evaluating drug efficacy and vaccine protection.

[0003] Mice are important models for studying the pathogenic mechanisms of pathogens and evaluating the protective effects of drugs and vaccines. However, as the natural host of Hantan virus (HTNV), mice typically exhibit latent infection without showing obvious disease symptoms. Currently, mouse models used for HTNV research can be mainly divided into four categories: conventionally immunocompetent mice, conventionally immunodeficient mice, transgenic immunocompetent mice, and transgenic immunodeficient mice.

[0004] Traditional immunocompetent mice mainly include Balb / c mice and newborn suckling mice. Balb / c mice are subclinically infected with HTNV, but because they produce high-titer antibodies, they are often used to evaluate vaccine-induced neutralizing antibody responses. Newborn suckling mice can die after HTNV infection, but their cause of death is mostly related to encephalitis, which is inconsistent with the clinical characteristics of human hemorrhagic fever with renal syndrome (HFRS). Therefore, this model is mainly used for virus amplification and some vaccine protective experiments. Traditional immunodeficient mice mainly include Nude mice and SCID mice. Both exhibit similar pathological manifestations after HTNV infection, including weight loss, and some mice even die, offering certain modeling advantages. However, this model requires approximately 20 days from infection to onset of symptoms, failing to simulate the acute progression of HFRS; furthermore, the symptoms in mice spontaneously resolve and gradually recover after about 35 days, exhibiting self-limiting characteristics, which significantly interferes with research on the pathogenesis of HTNV and drug efficacy evaluation. Regarding transgenic immunocompetent models, hu-NSC-A2 mice and Nlc3 mice have been reported. - / - Mice. The hu-NSC-A2 model is a humanized model constructed by introducing the human HLA-A2 gene into a NOD scid-gamma background (lacking T, B, and NK cells) and implanting artificial hematopoietic stem cells. Although this model can exhibit weight loss, lung inflammation, and human thrombocytopenia after HTNV infection, it lacks typical HFRS symptoms, and the survival period of artificial hematopoietic stem cells is limited (approximately 6 months), making model acquisition difficult. Nlc3 - / - The infection rate in mice was higher than that in wild-type mice, but they still did not exhibit HFRS symptoms. Transgenic immunodeficient mice mainly included type I interferon receptor-deficient A129 and MVAS. - / - Mice. A129 mice showed elevated viral load after infection, but no significant clinical symptoms; MVAS - / - Mice also did not show obvious symptoms. In summary, there is currently a lack of an ideal mouse disease model that can fully simulate the pathogenesis of HTNV and is suitable for mechanism research and efficacy evaluation. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a method for constructing and applying a mouse model of hemorrhagic fever with renal syndrome.

[0006] A method for constructing a mouse model of hemorrhagic fever with renal syndrome involves infecting mice with double deficiency of type I and type II interferon receptors with Hantan virus to obtain the mouse model of hemorrhagic fever with renal syndrome.

[0007] This invention, based on interferon receptor-deficient mice, successfully constructed a mouse disease model highly consistent with the clinical characteristics of human HFRS. All mice died within 9–15 days post-infection. Post-infection, the mice exhibited weight loss, hypothermia, multi-organ viral replication, and complete lethality, with symptoms mimicking human severe hemorrhagic fever with renal syndrome. Hantan virus replicated at high viral loads in the liver, spleen, lungs, and kidneys. The mouse model provided by this invention is completely lethal, with 10... 2 The viral load of FFU is lethal to mice. After mice are infected with HTNV, blood routine results show an increase in white blood cells and neutrophils, while lymphocytes show no significant changes, which is consistent with the blood routine changes of hemorrhagic fever patients.

[0008] Preferably, obtaining the type I and type II interferon receptor dual-deficient mice includes the steps of mating type I interferon receptor deficient mice with type II interferon receptor deficient mice or knocking out type I and type II interferon receptors in mice.

[0009] Preferably, when performing Hantan virus infection, the Hantan virus is strain 76-118.

[0010] Preferably, when performing Hantan virus infection, the route of infection is intramuscular injection, intraperitoneal injection, subcutaneous injection, or respiratory exposure.

[0011] Preferably, when performing Hantan virus infection, the titer of the Hantan virus is ≥1×10⁻⁶. 2 FFU / mL.

[0012] Preferably, the infection dose of the Hantan virus is 3×10² FFU / animal to 3×10² FFU / animal. 5 FFU / only.

[0013] Preferably, the mouse model of hemorrhagic fever with renal syndrome exhibits elevated white blood cell count, increased neutrophils, renal tubular necrosis, and thickening of the glomerular basement membrane.

[0014] Application of the mouse model of hemorrhagic fever with renal syndrome obtained by the above construction method in the study of the pathogenesis mechanism of Hantan virus.

[0015] Application of the mouse model of hemorrhagic fever with renal syndrome obtained by the above construction method in screening products for the treatment of Hantan virus infection.

[0016] The use of the mouse model of hemorrhagic fever with renal syndrome obtained by the above construction method in evaluating the protective efficacy of Hantan virus vaccine.

[0017] This invention successfully constructed a mouse disease model of HFRS that highly closely resembles the clinical characteristics of human HFRS, based on IFN receptor-deficient mice. All mice died within 9–15 days post-infection, exhibiting symptoms such as weight loss, hypothermia, multi-organ viral replication, and complete lethality, mimicking the symptoms of severe hemorrhagic fever with renal syndrome in humans. High viral loads of Hantan virus were observed in the liver, spleen, lungs, and kidneys. A systematic evaluation system was established by reviewing the model's disease progression, organ viral load, pathophysiological characteristics, and pathological damage. Subsequently, the effectiveness of the model in antibody therapy and vaccine protection was verified, and its key pathogenic mechanisms were preliminarily explored. This invention represents the first complete technical system from model construction and systematic evaluation to application verification and mechanism exploration, providing a novel experimental platform for research on the pathogenesis and prevention strategies of HFRS.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: The mouse model provided by this invention is completely lethal, 10 2 The viral load of FFU is lethal to mice. After mice are infected with HTNV, blood routine results show an increase in white blood cells and neutrophils, while lymphocytes show no significant changes, which is consistent with the blood routine changes of hemorrhagic fever patients.

[0019] Compared with wild-type C56 mice, type I interferon receptor-deficient mice, and type II interferon receptor-deficient mice, type I and type II interferon receptor knockout mice showed significantly increased viral load (protein and RNA levels) in the liver, spleen, lungs, and kidneys. Pathological sections showed obvious lesions in the liver, spleen, and lungs of the mice.

[0020] The results of antibody protection experiments showed that 2 mg / kg of 3G1 antibody could protect mice infected with HTNV from death, indicating that this model can be used for drug protection experiments. Attached Figure Description

[0021] Figure 1 This diagram illustrates the breeding and genotyping of AGB6 mice. In the diagram, A represents the process of obtaining AGB6, B represents the results of identifying the type I interferon receptor gene knockout in mice, and C represents the results of identifying the type II interferon receptor gene knockout in mice.

[0022] Figure 2 This study compares the susceptibility of three interferon receptor-deficient mice to HTNV infection. In this study, A represents the challenge strategies of the three interferon receptor-deficient mice, B represents weight change, C represents body temperature change, D represents the survival rate of mice after challenge, E represents the infection level of HTNV in the liver, F represents the infection level of HTNV in the spleen, G represents the infection level of HTNV in the lungs, and H represents the infection level of HTNV in the kidneys.

[0023] Figure 3, where A represents the sensitive lethal dose of HTNV in AGB6 mice, B represents the weight change curve of AGB6 mice after challenge, C represents the body temperature change of AGB6 mice after challenge, and D represents the survival curve of AGB6 mice after challenge.

[0024] Figure 4 The hematological dynamics of AGB6 mice induced by HTNV infection are shown in the figures. A represents the challenge strategy of AGB6 mice in the hematological dynamics experiment, B represents peripheral blood leukocytes, C represents neutrophil count, D represents monocyte count, E represents lymphocyte count, F represents neutrophil percentage, G represents monocyte percentage, and H represents lymphocyte percentage.

[0025] Figure 5 The replication kinetics and tissue tropism of HTNV in infected AGB6 mice are shown in Figure 1. A represents the dynamic transformation of HTNV nucleic acid in the liver, B represents the dynamic transformation of HTNV nucleic acid in the spleen, C represents the dynamic transformation of HTNV nucleic acid in the lungs, and D represents the dynamic transformation of HTNV nucleic acid in the kidneys.

[0026] Figure 6 This is a temporal analysis of pathological damage to major organs in AGB6 mice caused by HTNV infection. In this study, A represents the liver, B the spleen, C the lung, D the kidney, and E the electron microscopic observation results of kidney tissue.

[0027] Figure 7 To evaluate the protective efficacy of neutralizing antibody 3G1 against HTNV infection using the AGB6 mouse model, A represents the experimental design, B represents body temperature changes, C represents body weight changes, D represents survival analysis, E represents viral load in the liver, F represents viral load in the spleen, G represents viral load in the lungs, and H represents viral load in the kidneys. Detailed Implementation

[0028] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods.

[0029] I. Breeding diagram and genotyping of AGB6 mice 1. Mouse Breeding Strategy: Type 1 interferon receptor-deficient mice (AB6) with a C57BL / 6 background were purchased from Cyagen Biosciences Co., Ltd. Type 2 interferon receptor-deficient mice (GB6) with a C57BL / 6 background were purchased from Shanghai Southern Model Biotechnology Co., Ltd. AB6 and GB6 mice were mated to obtain heterozygous mice (F1 generation) with defects in both type I and type II interferon receptors. F1 generation mice were mated, and PCR was used to identify homozygous mice with defects in both type I and type II interferon receptors (AGB6).

[0030] 2. Mouse genotyping: (1) Collect mouse tails, add 200 μL of tissue DNA extraction solution A, boil in water for 30 min, then add 200 μL of tissue DNA extraction solution B, centrifuge at 6000 r / min for 3 min, and the supernatant is the liquid containing DNA, which is used as a template for mouse identification. The formula of tissue DNA extraction solution is shown in Table 1.

[0031] Table 1. Preparation method of tissue DNA extraction solution (2) Perform PCR on the samples. The DNA polymerase used is 2×Hieff® PCR Master Mix (10102ES03) from Yisheng Biotechnology. The primers, reaction system and PCR running procedure are shown in Table 2-4.

[0032] Table 2 Primers for PCR identification of mouse genotypes Table 3 PCR reaction system Table 4 PCR running procedure (3) Prepare a 1% agarose gel and electrophore the sample at 180V for 15 minutes, then take UV photos and count the results.

[0033] II. Comparison of susceptibility to HTNV infection in mice with three interferon receptor deficiencies 1. Virus source: HTNV (76-118 strain) was preserved by the Department of Microbiology and Pathogenic Biology, Air Force Medical University.

[0034] 2. Virus preservation and amplification: Vero E6 cells were infected with HTNV. After 1.5-2 hours, the medium was replaced with MEM medium containing 2% fetal bovine serum. The cells were cultured for 10-15 days and the samples were harvested. After repeated freeze-thaw cycles 3 times, the liquid was harvested and centrifuged at 4℃ and 5000rpm for 10 minutes. The harvested supernatant was the virus suspension.

[0035] 3. Virus titer determination: HTNV virus titer was detected by FFU. The relevant experimental method is referenced in: Ye C, et al. An Improved Enzyme-Linked Focus Formation Assay Revealed Baloxavir Acidas a Potential Antiviral Therapeutic Against Hantavirus Infection. FrontPharmacol. 2019 Oct 16;10:1203. doi: 10.3389 / fphar.2019.01203.

[0036] 4. Mouse challenge: Mice were divided into four groups: AB6 group, GB6 group, AGB6 group, and the control group (WT group, wild-type C57BL / 6 mice), with six mice in each group. The mice were challenged via intramuscular injection at a dose of 3.2 × 10⁻⁶. 5 FFU / mL. Mice were monitored daily for changes in body temperature, weight, and clinical signs. Mice were euthanized when they became ill and died, or when their weight dropped below 80% of their initial body weight. Liver, spleen, lung, and kidney tissue samples were harvested. Some tissues were fixed in 4% paraformaldehyde and sent to Chengdu Lilai Biotechnology Co., Ltd. for tissue embedding in paraffin blocks and HE staining. RNA samples were extracted from some tissues for RT-qPCR detection.

[0037] 5. RT-qPCR detection: Total RNA was extracted from tissues using the Tiangen Biotech Total RNA Extraction Kit (DP419). The RNA was reverse transcribed using the Novizan HiScript II Q RT SuperMix for qPCR Kit (R222-01), and RT-qPCR was performed using the Hieff UNICON® Universal Blue qPCR SYBR Green Master Mix Kit (11184ES03). Specific primer sequences, reaction systems, and operating procedures are shown in Tables 5-8.

[0038] 6. Generate a standard curve for the HTNV S gene: Use Takara's EASY Dilution reagent to perform 10-fold serial dilutions of the in vitro transcribed RNA (HTNV S gene) to obtain concentrations in the range of 1×10⁻⁶. 10 Up to 1×10 1Standard samples were prepared in copies / μL. The CT value of each dilution was detected by RT-qPCR, and a standard curve was generated using the CT value versus the logarithm of copies (log10 copies). The formula was: Y = (-3.3293)X + 34.73, where Y is the CT value and X is log10 (copy number). Primers and reaction systems used are shown in Tables 5-8. Method reference: Yang Q, et al. Establishment and optimization of arapid and convenient viral RNA transcript copy reduction neutralization test (VcRNT) for quantification of hantaan orthohantavirus (HTNV) neutralizing antibodies. Virology. 2025 Jul;608:110542. doi: 10.1016 / j.virol.2025.110542.

[0039] Table 5 RT-qPCR primers Table 6 RNA reverse transcription system Table 7 RT-qPCR reaction system Table 8 RT-qPCR Running Procedure III. Sensitive Lethal Dose of HTNV in AGB6 Mice Mouse challenge strategy: AGB6 mice were divided into 4 groups of 6 mice each. On day 0, the mice in each of the four groups were challenged with viral titers of 3 × 10⁻⁶ mcg / mL via intramuscular injection. 2 3×10 3 3×10 4 and 3×10 5 FFU / mL. The weight, body temperature, and survival status of mice were monitored and recorded daily after challenge.

[0040] IV. Hematological dynamics induced by HTNV infection in AGB6 mice 1. Mouse challenge strategy: Mice were divided into a control group (WT group, wild-type C57BL / 6) and an AGB6 group, with 30 mice in each group. The mice were challenged with 3 × 10⁻⁶ mice. 3Mice were injected intramuscularly with FFU / mL, and blood and tissue samples were collected on days 0, 3, 6, 9 and 12 post-infection.

[0041] 2. Complete blood count (CBC) test: Blood was collected from mice using Kangweishi disposable vacuum blood collection tubes (EDTAK2), and then randomly tested using a Mindray whole cell blood analyzer.

[0042] V. Replication kinetics and tissue tropism of HTNV in AGB6 infected mice The levels of HTNV S in various organs of mice were detected using RT-qPCR. The primers, standard curve, reagents, and operating procedures used were the same as before.

[0043] VI. Temporal analysis of pathological damage to major organs in AGB6 mice induced by HTNV infection 1. HE staining: The harvested mouse organ tissues were fixed by immersing them in 4% paraformaldehyde solution and then sent to Chengdu Lilai Biotechnology Co., Ltd. for tissue paraffin embedding and HE staining.

[0044] 2. Electron microscopy: Fresh mouse kidney tissue the size of a mung bean was taken, fixed in glutaraldehyde for 2 hours, then the tissue was trimmed and sent to the Electron Microscopy Center of the Department of Pathology, Basic Medical College, Air Force Medical University for electron microscopic examination.

[0045] VII. Evaluation of the protective efficacy of neutralizing antibody 3G1 against HTNV infection using the AGB6 mouse model 1. Mouse challenge strategy: One day before challenge, AGB6 mice were intraperitoneally injected with 3G1 antibody (2 mg, 10 mg, 25 mg) or PBS as a control; on day 0, mice were challenged by intramuscular injection (virus titer of 3 × 10³ FFU / mL), and their body weight, body temperature, and survival rate were monitored daily thereafter. The 3G1 antibody was prepared and preserved by the Department of Microbiology and Pathogenic Biology, Air Force Medical University.

[0046] 2. RT-qPCR was used to detect viral load in various organs of mice. The primers, standard curve, reagents, and operating procedures were the same as before.

[0047] Statistical Analysis: Experimental data were processed and statistically analyzed using GraphPad Prism 9.0 software. Results were plotted in a statistical format of mean ± standard deviation. One-way ANOVA was used for univariate analysis of data among multiple groups, followed by Tukey's multiple comparison test. Two-way ANOVA was used for two-way analysis of data among multiple groups, followed by Dunnett's t-test. p < 0.05 was considered statistically significant, * represents p < 0.05, ** represents p < 0.01, and *** represents p < 0.001.

[0048] result Figure 1 A schematic diagram of the breeding of AGB6 mice and genotyping identification. Figure 1 A illustrates the process of obtaining AGB6. Type I interferon receptor-deficient mice (AB6) were crossbred with type II interferon receptor-deficient mice (GB6) to obtain F1 generation mice, which are double heterozygous knockout mice of type I and type II interferon receptors. These F1 generation mice were then crossbred to select for AGB6 mice, which possess double homozygous knockout genes of type I and type II interferon receptors. Figure 1 B and Figure 1 C represents the AGB6 genotype identification result. Figure 1 B represents the results of identifying the knockout of the mouse type I interferon receptor gene using primers F1R1 and F1R2. When performing PCR using primer F1R1, WT mice produced a 2218 bp band, while AGB6 mice produced a 657 bp band. When performing PCR using primer F1R2, WT mice produced a 749 bp band, while AGB6 mice produced no specific band. Figure 1 C represents the results of knocking out the mouse type II interferon receptor gene using primers P1P2 and P3P4. When using primer P1P2 for PCR, WT mice produced a 1755 bp band, while AGB6 mice produced a 765 bp band. When using primer P3P4 for PCR, WT mice produced a 465 bp band, while AGB6 mice produced no specific band. Primers F1R1, F1R2, P1P2, and P3P4 are shown in Table 1.

[0049] Figure 2 This study compares the susceptibility of three interferon receptor-deficient mice to HTNV infection. Figure 2 A represents the challenge strategy for three interferon receptor-deficient mice. On day 0, mice were challenged via intramuscular injection at a dose of 3.2 × 10⁻⁶. 5 FFU / mL. The mice's body temperature, weight, and survival rate were then monitored daily. Except for mice that died naturally, mice were considered to be in an irreversible death process when their weight dropped below 80% of their original weight and were euthanized humanitarianally. After death, samples of major organs such as liver, spleen, lungs, and kidneys were harvested for subsequent experiments. Purple represents the WT group, red represents the AB6 group, green represents the GB6 group, and blue represents the AGB6 group. Figure 2 Figure B shows the changes in mouse body weight. As shown in Figure 2B, the body weight of mice in the WT combined GB6 group remained almost unchanged. The body weight of mice in the AB6 group gradually decreased after challenge, reaching its lowest point on day 8, after which it began to recover. The body weight of mice in the AGB6 group continued to decrease from day 5 after challenge until death. Figure 2 C represents the changes in mouse body temperature. For example... Figure 2As shown in Figure C, the body temperature of mice in the WT and GB6 groups remained relatively stable with no significant changes. The body temperature of mice in the AB6 group showed a brief increase on day 6, followed by a continuous decline, reaching its lowest point on day 10, after which the body temperature gradually recovered. The body temperature of mice in the AGB6 group began to decline continuously on day 7 after challenge, until death. Figure 2 D shows the survival rate of mice after challenge. No mice in the WT, AB6, and GB6 groups died after challenge, while mice in the AGB6 group began to die on day 10 and all died within 12 days. Figure 2 EH illustrates the detection of HTNV infection levels in major organs of mice in each group using RT-qPCR. The dashed line in the figure represents the limit of detection (LLOD). Results showed that no significant viral nucleic acid was detected in the liver, spleen, lung, and kidney tissues of mice in the WT and GB6 groups, indicating that HTNV failed to establish effective infection in these two mouse strains. In the AB6 group, high levels of viral nucleic acid were detected in the liver and spleen, while the viral load in the lungs and kidneys was relatively low, suggesting that although HTNV can infect AB6 mice, its replication ability is somewhat limited. Conversely, high viral nucleic acid loads were observed in all tested organs (liver, spleen, lung, and kidney) of the AGB6 group mice, indicating that HTNV can replicate extensively in this mouse strain.

[0050] Figure 3 The sensitive lethal dose of HTNV in AGB6 mice. Figure 3 A represents the mouse challenge strategy for this experiment: On day 0, mice in four groups were challenged with intramuscular injections of the virus, with viral titers of 3 × 10⁻⁶ and 10⁻⁶ respectively. 2 3×10 3 3×10 4 and 3×10 5 FFU. Daily monitoring and recording of mouse weight, body temperature, and survival status were conducted after challenge. In the figure, purple, red, green, and blue represent different virus dosage groups (from highest to lowest). Figure 3 B shows the body weight change curve of AGB6 mice after challenge. The results showed that all groups experienced weight loss 5-6 days after infection, with the 3×10 group showing the largest decrease. 5 FFU (purple) and 3×10 4 The FFU group (red) experienced a faster rate of weight loss, while the 3×10 3 FFU group (green) and 3×10 2 The FFU group (blue) showed a slower rate of weight loss. These results indicate that the higher the challenge dose, the faster the body weight of AGB6 mice decreased after infection, exhibiting a clear dose-dependent effect. Figure 3 C shows the changes in body temperature in AGB6 mice after challenge. All groups showed a decrease in body temperature, but the timing of this decrease differed in a dose-dependent manner: 3 × 10⁻⁶. 5and 3×10 4 The FFU group showed a decrease in body temperature on day 5, while the 3×10 3 and 3×10 2 The FFU groups showed delayed onset of fever on day 8 and day 13, respectively. The results indicated that a higher dose of the toxic agent led to an earlier onset of fever decline in mice, suggesting more rapid disease progression. Figure 3 D represents the survival curves of AGB6 mice after challenge with the virus. As shown in the figure, all challenged mice eventually died, but the time of death was dose-dependent. Specifically, 3 × 10⁻⁶ mice... 5 and 3×10 4 All mice in the FFU group died on day 9, while 3×10 3 and 3×10 2 The time to death of all mice in the FFU group was delayed, occurring on day 12 and day 15, respectively. These results indicate that 3 × 10 2 FFU doses of HTNV can cause death in AGB6 mice.

[0051] Figure 4 The dynamic changes in hematology induced by HTNV infection in AGB6 mice. Figure 4 A demonstrates the challenge strategy for AGB6 mice in this experiment: On day 0, mice in both the WT group and the AGB6 group were challenged with intramuscular injection of 3 × 10⁻⁶ mol / L virus at a dose of 3 × 10⁻⁶. 3 FFU / mL. Blood and tissue samples were collected on days 0, 3, 6, 9, and 12 for testing. The WT group is represented by purple, and the AGB6 group is represented by blue. Figure 4 BG represents the dynamic changes in blood routine indicators at different time points in mice after infection with HTNV. Figure 4 As shown in Figure B, on day 9 post-infection, the total peripheral blood leukocyte count in AGB6 mice was significantly elevated, consistent with the clinical characteristics of HFRS patients. Leukocyte differential count showed a particularly pronounced increase in neutrophil count. Figure 4 B, E), while no significant changes were observed in the number of monocytes and lymphocytes (B, E), Figure 4 C, F and Figure 4 D, G). This inflammatory response pattern, characterized primarily by neutrophilia, further mimics the typical blood routine characteristics of HFRS patients.

[0052] Figure 5 To investigate the replication kinetics and tissue tropism of HTNV in AGB6 infected mice. Figure 5The AD study demonstrates the dynamic changes in HTNV nucleic acid in the liver, spleen, lungs, and kidneys of AGB6 mice after infection with HTNV, detected by RT-qPCR. The results showed that viral nucleic acid levels in the major organs began to rise 3 days post-infection, peaking on day 6, and remained at a high level until the end of the observation period (day 12). These results indicate that HTNV can rapidly replicate and establish widespread, persistent infection in AGB8 mice.

[0053] Figure 6 The temporal progression of multi-organ pathological damage induced by HTNV infection in AGB6 mice was analyzed. As shown in the figure, all major organs exhibited characteristic and progressively worsening pathological changes. Liver ( Figure 6 A) Pericentral venous inflammatory cell infiltration appeared on day 6 post-infection, worsened and was accompanied by bleeding on day 9, and significant hepatocellular necrosis was observed by day 12. Spleen ( Figure 6 B) In the later stages of infection (days 9 and 12), extensive proliferation of white pulp occurs, blurring its boundary with the red pulp, and reducing the number of red blood cells within the red pulp. Lungs ( Figure 6 C) Peribronchial inflammatory cell infiltration and alveolar edema appeared on day 9, and the damage worsened further on day 12. Kidneys ( Figure 6 The lesions in D) were particularly typical. On day 12 of infection, renal interstitial hemorrhage, necrosis and shedding of renal tubular epithelial cells, and formation of cellular casts were observed. Poor opening of glomerular capillary loops was observed, with neutrophils and erythrocytes visible within the lumen, and some endothelial cells swollen, leading to luminal narrowing. Figure E shows the electron microscopic observation of kidney tissue in mice 12 days after infection. Numerous viral particles and high electron density protein deposits were observed in the cytoplasm of glomerular mesangial cells (first and second columns); the glomerular basement membrane was uneven in thickness, showing an overall thickening trend, with a maximum thickness reaching 425 nm (third column); a large number of necrotic cell fragments were visible in the renal tubulointerstitium (fourth column). These results collectively indicate that the infection severely damaged the kidney's filtration barrier (glomerulus) and reabsorption functional unit (tubulointerstitium). In conclusion, HTNV infection can cause multi-organ damage in AGB6 mice, especially mimicking key renal pathological manifestations in the course of human HFRS, indicating that this model is a reliable tool for studying the pathogenesis of hemorrhagic fever with renal syndrome.

[0054] Figure 7 The protective efficacy of neutralizing antibody 3G1 against HTNV infection was evaluated using the AGB6 mouse model. Figure 7A is a schematic diagram of the experimental design: One day before challenge, AGB6 mice were intraperitoneally injected with 3G1 antibody (2 mg, 10 mg, 25 mg) or PBS as a control; on day 0, they were challenged by intramuscular injection (viral titer of 3 × 10³ FFU / mL), and the mice's body weight, body temperature, and survival rate were monitored daily thereafter. In the figure, purple, red, green, and blue represent the PBS group, 2 mg group, 10 mg group, and 25 mg antibody group, respectively. The results showed that mice in the PBS group experienced a decrease in body temperature on day 9 post-infection (…). Figure 7 B), while the body temperature of mice in each antibody group remained normal. Regarding weight changes, the weight of mice in the PBS group decreased significantly from day 5 onwards, while the weight of mice in the antibody group remained stable or slightly increased. Figure 7 C) indicates that antibody treatment effectively alleviated the physiological disturbances caused by the infection. Survival analysis showed that all mice in the PBS group died, while no deaths occurred in any of the antibody groups (C). Figure 7 (D) further demonstrates that the 3G1 antibody provides complete protection against HTNV infection. Furthermore, the viral load in major organs was detected by RT-qPCR ( Figure 7 (EH) No significant viral replication was detected in any tissue of the antibody-treated mice, significantly lower than in the PBS group. In conclusion, the 3G1 antibody effectively protects AGB6 mice against HTNV challenge, preventing viral replication and lethal infection, demonstrating that AGB6 mice can serve as a reliable animal model for evaluating the protective effect of therapeutic antibodies against HFRS.

[0055] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0056] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0057] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A method for constructing a mouse model of hemorrhagic fever with renal syndrome, characterized in that, The mouse model of hemorrhagic fever with renal syndrome was obtained by infecting mice with dual deficiency of type I and type II interferon receptors with Hantan virus.

2. The construction method according to claim 1, characterized in that, Obtaining mice with dual deficiency of type I and type II interferon receptors includes the steps of mating type I interferon receptor-deficient mice with type II interferon receptor-deficient mice or knocking out type I and type II interferon receptors in mice.

3. The construction method according to claim 1, characterized in that, When Hantan virus infection was performed, the Hantan virus strain was 76-118.

4. The construction method according to claim 1, characterized in that, When Hantan virus infection occurs, the route of infection is intramuscular injection, intraperitoneal injection, subcutaneous injection, or respiratory exposure.

5. The construction method according to claim 1, characterized in that, When performing Hantan virus infection, the titer of the Hantan virus must be ≥1×10⁻⁶. 2 FFU / mL.

6. The construction method according to claim 1, characterized in that, The mouse model of hemorrhagic fever with renal syndrome exhibited elevated white blood cell count, increased neutrophils, renal tubular necrosis, and thickening of the glomerular basement membrane.

7. The application of the mouse model of hemorrhagic fever with renal syndrome obtained by the construction method according to any one of claims 1-6 in the study of the pathogenesis or immune escape mechanism of Hantan virus.

8. The application of the mouse model of hemorrhagic fever with renal syndrome obtained by the construction method according to any one of claims 1-6 in screening products for the treatment of Hantan virus infection.

9. The use of the mouse model of hemorrhagic fever with renal syndrome obtained by the construction method according to any one of claims 1-6 in evaluating the protective efficacy of Hantan virus vaccine.

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