Lassa fever suckling mouse infection model as well as establishment method and application thereof
By constructing a recombinant VSV-LASV virus to simulate the neural invasion process of Lassa virus in a BALB/c suckling mouse model, the problem of studying Lassa fever under low-protection laboratory conditions was solved, and safe and efficient virus research and drug screening were achieved in a BSL-2 laboratory.
Patent Information
- Application Number
- CN202511213822.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-28
- Publication Date
- 2025-12-05
AI Technical Summary
The lack of alternative models for studying Lassa fever virus under low-protection-level laboratory conditions leads to resource constraints and operational risks in biosafety level 4 laboratories for vaccine and drug development, and the problem of antigenic variation caused by viral genetic diversity has not been effectively solved.
Based on the VSV reverse genetics platform, a recombinant chimeric virus VSV-LASV expressing Lassa virus glycoprotein was constructed to establish a lethal infection model in a biosafety level 2 laboratory. The virus was injected intraperitoneally into 3-day-old BALB/c suckling mice to simulate the process of viral invasion of host cells and its key neutralizing epitope characteristics.
While lowering research thresholds and operational risks, it significantly accelerates the development of antiviral drugs and vaccines, provides an alternative research tool for Lassa virus under BSL-2 conditions, can simulate the neural invasion and pathogenic mechanisms of the virus, and evaluate the in vivo protective effect of neutralizing antibodies.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the field of biotechnology, and provides a Lassa fever mouse infection model suitable for a biosafety level 2 (BSL-2) laboratory and a method for establishing the same and applications thereof. BACKGROUND
[0002] Lassa mammarenavirus (LASV) is a member of the Old World virus of the Arenaviridae family and the causative agent of Lassa fever, an acute febrile illness endemic in West Africa. LASV is an enveloped, bipartite, single-stranded RNA virus that encodes four viral genes using an ambisense coding strategy. Based on the spill-over risk model, the annual infection amount in the region can reach 900,000 cases, and the virus has caused a significant public health burden, but there is still a lack of approved vaccines and effective treatment options. The only nucleoside analogue with therapeutic value, ribavirin, has serious side effects and only shows efficacy when administered in the early stage of infection. Since the pathogen LASV needs to be operated in a biosafety level 4 (BSL-4) laboratory, and it is difficult to obtain clinical samples, the pathophysiological mechanisms of severe cases have not been clarified so far. The control of the disease depends on the rapid balance between the immune response and viral clearance, and the characteristics of severe cases are the excessive activation of innate immunity, which leads to symptoms similar to sepsis and a cytokine storm. It is estimated that 9% of the population in the epidemic area has been exposed to the virus, and it is predicted that the epidemic area will expand in the coming decades, putting more and more people at risk of viral infection and disease transmission. Therefore, Lassa fever has been included in the list of priority diseases that need to be urgently addressed by the WHO.
[0003] Lassa fever caused by Lassa virus is a severe infectious disease characterized by acute fever, systemic bleeding tendency, and multiple organ failure, and is endemic in West Africa, with a mortality rate of 15%-30%. It is worth noting that, although there are similar mechanisms of vascular damage and immunopathology (such as endothelial cell dysfunction and uncontrolled release of inflammatory factors) as Ebola virus, Lassa fever currently only has ribavirin as a limited efficacy treatment drug, and there is no internationally widely used preventive vaccine. Although candidate vaccines based on viral vectors (such as VSV, MOPV) have entered the clinical trial stage, the problem of antigen variation and unstable immunogenicity caused by viral genetic diversity is still a core obstacle to vaccine development. Given that the virus is listed as a priority pathogen by the World Health Organization and has the potential for aerosol transmission, its prevention and control research not only involves disease treatment, but also relates to the construction of biological defense systems.
[0004] The core difficulty in the study of Lassa virus lies in the strict operation restrictions of its biosafety level 4 (BSL-4) pathogen, and the related experiments are highly dependent on professional protection facilities and long-term financial support. The development of vaccines and drugs faces multiple challenges: the lack of local virus strain library, the serious dependence on imported high-level animal models such as non-human primates, and the difficulty of BSL-4 laboratory resources to meet the large-scale research needs. Therefore, it is crucial to develop an alternative technology system that can be safely operated in a low protection level laboratory, including the construction of a pseudovirus evaluation platform expressing Lassa virus glycoprotein, and the establishment of a new rodent model with human pathological characteristics (such as humanized mice, Syrian golden hamsters, and other adaptive infection models). These breakthroughs not only accelerate the development process of antiviral drugs and vaccines, but also provide key technical support for joint prevention and control of emerging epidemics.
[0005] Vesicular Stomatitis Virus (VSV) has become a key technology platform for constructing pathogen model viruses based on reverse genetics due to its highly plastic genome, low biosafety risk (complying with BSL-2 level), and efficient expression of foreign genes. By chimeric glycoprotein (GP) of the target virus (such as Lassa virus LASV) into the VSV backbone, the constructed chimeric virus can accurately simulate the process of wild virus invading host cells and its key neutralizing epitope characteristics, not only reproducing the receptor binding and membrane fusion mechanism dependent on natural viruses, but also widely applicable to high-throughput detection of neutralizing antibodies, evaluation of immunogenicity of vaccine candidates, and research on cross-species transmission mechanism. Under the condition of lacking high-level biosafety laboratory (such as BSL-3, BSL-4), this technology system provides a core alternative solution for basic research of high-risk pathogens such as Lassa virus, supporting antibody efficacy evaluation and animal infection model construction without operating live virus, significantly accelerating the process of antiviral drug screening and new vaccine development.
[0006] Based on this, the present application intends to develop a Lassa virus alternative animal infection model suitable for BSL-2 laboratory environment, with operational economy and experimental efficiency. SUMMARY
[0007] Based on the VSV reverse genetics technology platform, the present application successfully constructs a recombinant Vesicular Stomatitis Virus (VSV-LASV) expressing Lassa virus glycoprotein (GP), and establishes a lethal infection model on 3-day-old BALB / c mice using the recombinant virus. The core breakthrough of this model lies in that the entire operation process (including virus propagation, animal infection, and sample detection) can be safely conducted in a standard biosafety level 2 (BSL-2) laboratory, which completely overcomes the strict dependence of wild-type Lassa virus (BSL-4) research on high-level biosafety laboratories, significantly reducing the research threshold, operation risk, and facility cost.
[0008] In a first aspect, the present application provides a method for establishing a surrogate infant mouse model of Lassa fever, which comprises infecting 3-day-old BALB / c infant mice with a recombinant chimeric virus VSV-LASV at a dose of 10 3.5 TCID 50 via intraperitoneal injection; wherein the recombinant chimeric virus VSV-LASV is a vesicular stomatitis virus (VSV) vector constructed by reverse genetics technology, in which the envelope glycoprotein GP gene in the genome is completely replaced by the glycoprotein (GP) gene of Lassa virus (LASV) Josiah strain, thereby chimerically expressing the LASV GP protein.
[0009] In particular, the application comprises the following steps: replacing the G gene in the VSV infectious clone full-length plasmid carrying a green fluorescent protein (GFP) reporter gene tag with the GP gene of Lassa virus Josiah strain to obtain a recombinant full-length plasmid pVSV-ΔG-GFP-LASV GP; co-transfecting the full-length plasmid with four auxiliary plasmids pVSV-N, pVSVS-P, pVSVS-L and pVSV-G expressing VSV nucleocapsid proteins into BSR cells; rescuing and obtaining the recombinant chimeric virus VSV-LASV carrying a GFP tag or a Luc tag and expressing the LASV GP.
[0010] In a second aspect, the present application provides a surrogate infant mouse model of Lassa fever obtained by the above method.
[0011] In a third aspect, the present application provides the use of the above-mentioned surrogate infant mouse model of Lassa fever in an experimental environment with a biosafety level of two (BSL-2) or lower.
[0012] In particular, the use comprises the study of Lassa virus glycoprotein (GP)-mediated neural invasion and pathogenic mechanisms, and the evaluation of the in vivo protective effect of Lassa virus neutralizing antibodies, antiviral drugs or vaccines.
[0013] In particular, the use comprises the study of Lassa virus glycoprotein (GP)-mediated neural invasion and pathogenic mechanisms, and the evaluation of the in vivo protective effect of Lassa virus neutralizing antibodies, antiviral drugs or vaccines.
[0014] The 3-day-old BALB / c suckling mice selected in the experiment have multiple advantages. The small size (about 2-3 g) greatly increases the number of animals accommodated in a unit of feeding space, effectively reduces the space load of the BSL-2 laboratory and reduces the risk of operation exposure. At the same time, this size is very suitable for implementing precise standardized intraperitoneal injection, thereby significantly improving the repeatability and operation safety of the experiment. In addition, compared with non-human primate models, the extremely low cost of the suckling mice brings extremely high cost-effectiveness, making large-scale challenge protection experiments, drug screening and vaccine evaluation a reality.
[0015] After being challenged by VSV-LASV, the model mainly presents significant infection characteristics in the central nervous system (brain) and causes death, which can effectively simulate the pathological process of potential virus breakthrough of the blood-brain barrier and invasion of the central nervous system in wild-type Lassa virus infection. Compared with the golden hamster model mainly targeting liver and spleen, this model exhibits unique and significant advantages in simulating the neurotropism and pathogenesis of Lassa virus and evaluating the in vivo effects of antiviral drugs, especially drugs that need to evaluate their delivery efficiency to the central nervous system. At the same time, combined with a small animal live imaging system, real-time, dynamic and non-invasive in vivo visualization monitoring and tracking of virus infection can be achieved.
[0016] In summary, the safe (BSL-2 operation), economical, easy-to-operate and key neuro-pathological characteristic-simulating alternative model provided by the present application provides a powerful standardized tool for in-depth study of Lassa virus pathogenesis (especially neural mechanisms), efficient evaluation of anti-Lassa virus antibodies, vaccines and antiviral drugs (including CNS delivery efficiency), effectively breaking through the biosafety bottleneck of high pathogenic virus research and accelerating the development of emergency medical countermeasures. BRIEF DESCRIPTION OF DRAWINGS
[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments described in the present application, and other drawings can also be obtained by those skilled in the art based on these drawings.
[0018] Figure 1 The structural mode diagram of the LASV Josiah strain recombinant VSV full-length plasmid provided by the embodiments of the present application.
[0019] Figure 2 The VSV-LASV recombinant chimeric virus rescue mode diagram provided by the embodiments of the present application.
[0020] Figure 3 The morphological observation results of the recombinant chimeric virus VSV-LASV under a transmission electron microscope provided by the embodiments of the present application.
[0021] Figure 4 Survival rate and body weight change of BALB / c mice of different ages infected by VSV-LASV provided by the embodiments of the present application.
[0022] Figure 5 LD50 of 3-day-old BALB / c mice infected by VSV-LASV provided by the embodiments of the present application. 50 Determination.
[0023] Figure 6 Viral load in tissues of 3-day-old BALB / c mice at different time points infected by VSV-LASV provided by the embodiments of the present application.
[0024] Figure 7 Dynamic monitoring of virus tropism in 3-day-old BALB / c mice infected by VSV-LASV provided by the embodiments of the present application.
[0025] Figure 8 Survival rate of 3-day-old BALB / c mice infected by VSV-LASV and other recombinant VSV recombinant viruses provided by the embodiments of the present application.
[0026] Figure 9 Survival rate and body weight change of 3-day-old BALB / c mice infected by VSV-LASV VIII / IV / VII recombinant viruses provided by the embodiments of the present application.
[0027] Survival rate and body weight change of 3-day-old BALB / c mice infected by VSV-LASV VIII / IV / VII recombinant viruses provided by the embodiments of the present application.
[0028] Figure 10 Survival rate of various adult rodents and mice infected by recombinant chimeric virus VSV-LASV provided by the embodiments of the present application. DETAILED DESCRIPTION
[0029] In order for those skilled in the art to better understand the technical solutions of the present application, the present application will be further described in detail below in combination with embodiments and drawings. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.
[0030] The test materials used in the following embodiments are commercially available from conventional biochemical reagent stores, unless otherwise specified. The experimental methods in the following embodiments are conventional methods, unless otherwise specified.
[0031] Example 1 Construction and identification of recombinant chimeric virus (VSV-LASV) expressing glycoprotein of Lassa virus
[0032] 1.1 Rescue and subculture of recombinant virus
[0033] To construct a Lassa fever surrogate virus that can be manipulated in a biosafety level two (BSL-2) laboratory, the present invention is based on the modification of the vesicular stomatitis virus (VSV) reverse genetics system. The core strategy is to replace the VSV envelope glycoprotein G with the LASV GP protein, so that the recombinant virus acquires the cell tropism characteristics of LASV, while retaining the safety and replication ability of the VSV backbone. (The Lassa virus strain is the Josiah strain, and both VSV and LASV are preserved by the Military Veterinary Institute)
[0034] Specific steps:
[0035] 1. Molecular cloning construction: using the full-length plasmid of VSV infectious clone carrying the GFP reporter gene (pVSV-GFP) as the backbone, the GP gene is precisely replaced with the GP gene of Lassa virus (LASV) Josiah strain by restriction enzyme digestion-ligation technology, to obtain the recombinant full-length plasmid pVSV-ΔG-GFP-LASV GP. Figure 1 (Note: Josiah strain is a standard virulent strain of LASV, and the GP-mediated receptor binding and membrane fusion function is the key to pathogenicity)
[0036] 2. Virus rescue: four kinds of helper plasmids are co-transfected to rescue the system. Figure 2 The recombinant full-length plasmid pVSV-ΔG-GFP-LASV GP and the helper plasmids pVSV-N, pVSV-P, pVSV-L and pVSV-G expressing VSV core proteins are mixed according to the standard ratio. BSR cells pre-cultured to 80% confluence in a six-well plate are transfected using calcium phosphate transfection reagent. 16-18 hours after transfection, treat with 10% DMSO in PBS solution for 2.5 minutes (shock treatment to improve efficiency), and replace with 2% fetal bovine serum (FBS) in DMEM maintenance medium for continuous culture.
[0037] 3. Virus harvesting and amplification: 48-72 hours after transfection, when obvious cytopathic effect (CPE, such as cell rounding and shedding) is observed, collect the cell culture supernatant, release the virus particles by three freeze-thaw cycles, centrifuge to remove cell debris to obtain the primary rescued virus. Virus supernatant is inoculated into fresh Vero E6 cells (sensitive to VSV and LASV GP), and after adsorption at 37°C, 5% CO2 for 1 hour, replace with 2% FBS in DMEM maintenance culture. Observe CPE and harvest supernatant every 48-72 hours, and continuously pass 5 times.
[0038] 4. Results and stability verification: During the passaging process on Vero E6 cells, stable and reproducible CPE was observed from P1 generation, which manifested as rapid rounding, clustering into grape-like clusters and finally shedding of the cells. The P5 generation virus supernatant was aliquoted and stored at -80°C for later use. This result confirmed the successful rescue of a recombinant virus with stable replication capacity, designated as rVSVAG-GFP-LASV GP (abbreviated as VSV-LASV).
[0039] 1.2 Transmission electron microscopy (TEM) morphological identification of recombinant virus
[0040] To evaluate the expression of LASV GP on the surface of recombinant virus particles and its impact on virus morphology, and to verify its structural basis as a surrogate virus for LASV:
[0041] 100 μL of P5 generation VSV-LASV virus supernatant was concentrated and purified by ultracentrifugation (e.g. 100,000 g, 2 hours). The precipitate was resuspended in a small amount of PBS buffer (pH 7.4). 10 μL of the virus suspension was added dropwise to a copper grid covered with carbon film, and after 1 minute of adsorption, the excess liquid was absorbed with filter paper. 2% (w / v) phosphotungstic acid (PTA) aqueous solution (pH 6.8) was added dropwise for negative staining for 1 minute, then dried. Transmission electron microscopy was used to observe and photograph at an acceleration voltage of 80 kV.
[0042] The results show that Figure 3 ), the electron microscopy images clearly showed that the recombinant virus VSV-LASV particles exhibited a typical VSV bullet / rod-shaped morphology (length about 150-180 nm, diameter about 70 nm), with an intact capsid structure, consistent with the morphological characteristics of wild-type VSV particles. The viral particle envelope surface showed uniformly distributed dense filamentous structures about 8-10 nm in length.
[0043] Example 2 Establishment of VSV-LASV infection model
[0044] 2.1 Study of the lethality of VSV-LASV in BALB / c suckling mice
[0045] To evaluate the pathogenicity of recombinant virus VSV-LASV in vivo and its relevance to the developmental stage of the host, this study systematically analyzed the infection outcomes of the virus in BALB / c suckling mice of different ages. Two groups of infection were set up: one group of 3-day-old mice (P3 group) and another group of 5-day-old mice (P5 group), both of which were inoculated with 10 3.5 TCID 50VSV-LASV. Three-day-old (Uninfected P3 group) and five-day-old (Uninfected P5 group) mice were injected i.p. with equal volume of PBS as controls. All animals were housed under BSL-2 laboratory conditions, and monitored daily for survival status, body weight change, and specific neurological symptoms including tremor, ataxia, limb paralysis, convulsion, and so on, as well as activity, coat smoothness, and suckling.
[0046] The experimental results revealed a significant age-dependent lethality. P5 group infected mice all survived to the end of the experiment (15 dpi), with body weight growth curves essentially overlapping with the Uninfected P5 control group ( Figure 4 ), and without any clinical symptoms. In sharp contrast, P3 group infected mice exhibited clear lethality ( Figure 4 ). Early after infection (1-4 dpi), P3 group body weight growth was similar to the Uninfected P3 control group (about 50% increase). However, from 5 dpi, infected mice began to show progressively aggravated neurological symptoms (e.g., reduced activity, tremor, hind limb weakness), accompanied by a sharp decrease in body weight ( Figure 4 ). Death events were concentrated in 6-11 dpi, eventually leading to all deaths. The control group (Uninfected P3) all survived healthily to the predetermined endpoint (15 dpi), with continuous body weight growth.
[0047] 2.2 Characteristics of VSV-LASV infection in 3-day-old BALB / c mice
[0048] To further analyze the lethal mechanism of P3 mice and its potential link to the neuroinvasiveness of wild-type Lassa fever, we further carried out key verification experiments. First, we determined the median lethal dose (LD 50 ) of VSV-LASV in 3-day-old mice infected i.p. by the Reed-Muench method, and designed eight groups of 3-day-old BALB / c mice infected with different doses of VSV-LASV (10 0.05 TCID 50 , 10 0.5 TCID 50 , 10 1.5 TCID 50 , 10 2.5 TCID 50 , 10 3.5 TCID 50 , 10 4.5 TCID 50 , 10 5.5 TCID 50 , 10 6.5 TCID 50), of which 10 2.5-6.5 TCID 50 The mortality rate of the infected suckling mice in the six groups was 100%. 1.5 TCID 50 The mortality rate of infected suckling mice was 92%, 10 0.5 TCID 50 The mortality rate of infected suckling mice was 60%, 10 0.05 TCID 50 The mortality rate of infected suckling mice was 0%. Simultaneously, infected suckling mice experienced slow weight gain, and 10 2.5-6.5 TCID 50 Infected suckling mice began to lose weight around 5 days post-infection (dpi), while all uninfected control mice survived and gained weight. Figure 5 As shown. The result is 10. 0.48 TCID 50 This confirmed that the model possesses stable and quantifiable pathogenicity. To further investigate VSV-LASV replication in 3-day-old BALB / c suckling mice, multiple tissues and organs, including the heart, liver, spleen, lung, kidney, brain, and intestine, were collected from euthanized P3 suckling mice at different time points (e.g., 0, 2, 4, 6, 8 dpi) to detect TCID viral load in these tissues. 50 Although viral replication can be detected in the gut at an early stage (with titers reaching up to 10 in the gut), it has been found that viral replication can be detected in the gut at an early stage. 7.3 TCID 50 / g), but the viral load showed explosive growth in brain tissue of the central nervous system (CNS) (peaking at 10 g). 8.22 TCID 50 The viral load ( / g) was significantly and persistently higher than in all peripheral organs, and the virus was undetectable in uninfected control tissues. Figure 6 As shown.
[0049] In addition, small animal in vivo imaging was used to dynamically monitor VSV-LASV in 3-day-old suckling mice. Two infection groups were set up, with 3 mice in each group (repeated): one group was the control group (con 1-3), and the other group was the VSV-LASV infection group (LASV 1-3). Both groups were inoculated via standardized intraperitoneal injection. 3.5 TCID 50Dose of VSV-LASV was given to examine the tropism and distribution of VSV-LASV in 3-day-old BALB / c suckling mice at different time points. The results showed that no viral signal was detected in the control group, while the viral signal intensity in the VSV-LASV infected group increased exponentially with the progression of the disease. At the beginning, the virus only existed in the abdominal cavity and gradually increased with the progression of the disease. It was visually captured that after 3 dpi, the fluorescence signal began to specifically enrich in the brain region of individual mice and continued to strengthen (as shown in Figure 7 The distribution pattern was consistent with the results of tissue viral load analysis, providing direct evidence of non-invasive visualization for the virus to break through the blood-brain barrier and target the CNS.
[0050] P3 infected mice showed characteristic biphasic course (early asymptomatic weight gain, late sudden onset of neurological symptoms leading to death) and clear central nervous tropism (viral load in brain tissue was significantly the highest), which highly mimicked the pathological process of encephalitis / encephalomyelitis complications reported in human cases of severe Lassa fever. This model, which is distinguished from the core feature of the golden hamster model mainly with liver and spleen damage, is specifically neurotargeted, making it a unique and powerful tool for studying the mechanisms of neural invasion and pathogenesis (such as blood-brain barrier crossing mechanism) mediated by Lassa virus (or its glycoprotein) under BSL-2 conditions, as well as evaluating the in vivo effects of antiviral drugs (especially neuroactive drugs that need to evaluate their CNS delivery efficiency).
[0051] 2.3 Glycoprotein-related study of VSV-LASV infection in 3-day-old BALB / c suckling mice
[0052] To discuss the correlation between the glycoprotein in the recombinant virus and the lethal phenomenon after VSV-LASV infection in 3-day-old mice, this study selected VSV-LASV and five other recombinant viruses (vesicular stomatitis virus recombinant Indiana strain (VSV-eGFP), vesicular stomatitis virus recombinant Reston virus (VSV-Reston-GP), vesicular stomatitis virus recombinant RAVN virus (VSV-Ravn-GP), vesicular stomatitis virus recombinant Middle East respiratory syndrome coronavirus Al-Hasa_1_2013 isolate (VSV-MERS), and vesicular stomatitis virus recombinant novel coronavirus 2 (VSV-SARS CoV2-S), which were constructed and preserved by the Animal Virology and Special Animal Disease Laboratory of the Changchun Animal Husbandry Institute, Chinese Academy of Agricultural Sciences) to infect 3-day-old BALB / c mice, each of which was inoculated with 10 3.5 TCID 50The six recombinant viruses were constructed by replacing the G gene of the VSV infectious clone full-length plasmid carrying the GFP or eGFP reporter gene tag with its GP gene or S gene. The recombinant full-length plasmid was co-transfected with four helper plasmids pVSV-N, pVSVS-P, pVSVS-L, and pVSV-G expressing the VSV nucleocapsid protein into BSR cells. The recombinant virus carrying the GFP tag was rescued and amplified. Three-day-old mice (Uninfected) were injected intraperitoneally with an equal volume of PBS as a control group. All animals were raised in a BSL-2 laboratory, and their survival status and specific neurological symptoms including tremors, ataxia, limb paralysis, convulsions, and the like were closely monitored daily. At the same time, the activity, fur, and nursing of the animals were observed.
[0053] Results: Only the 3-day-old BALB / c mice in the VSV-LASV infection group died, while the rest of the groups did not die (as shown in Figure 8 ). This indicates that the LASV GP is a key virulence factor that causes fatal infection in 3-day-old BALB / c mice.
[0054] 2.4 Strain lineage specificity study of VSV-LASV infection in 3-day-old BALB / c mice
[0055] To discuss the specificity of mouse death caused by VSV-LASV infection in this study, which is related to the LASV strain. In this study, three recombinant viruses of VSV-LASV IV Josiah, VSV-LASV III Nig-Ga391, and VSV-LASV VI Togo (VSV-LASV III Nig-Ga391 strain and VSV-LASV VII Togo strain were constructed and preserved by the Animal Virology and Special Animal Disease Laboratory of the Changchun Veterinary Research Institute of the Chinese Academy of Agricultural Sciences) were selected, each of which was inoculated with the highest titer of 10 5.5 TCID 50 Three-day-old mice (Uninfected) were injected intraperitoneally with an equal volume of PBS as a control group. All animals were raised in a BSL-2 laboratory, and their survival status and specific neurological symptoms including tremors, ataxia, limb paralysis, convulsions, and the like were closely monitored daily. At the same time, the activity, fur, and nursing of the animals were observed.
[0056] Results: Only the 3-day-old BALB / c mice in the VSV-LASV IV Josiah infection group died, while the rest of the groups did not die, and the body weight of the rest of the VSV-LASV infection groups showed an upward trend (as shown in Figure 9The pathogenicity of LASV was shown to be significantly strain lineage-specific. Glycoprotein (GP) of the IV lineage (Josiah strain) was the key virulence factor that caused lethal infection in 3-day-old BALB / c suckling mice, while GPs of the III and VII lineages did not exhibit lethality in the present model.
[0057] 2.5 Age and species specificity of pathogenicity of VSV-LASV recombinant virus
[0058] To evaluate the pathogenicity of the recombinant virus VSV-LASV in vivo and its correlation with the developmental stage and species of the host, 3-day-old BALB / c suckling mice, adult BALB / c mice, SD rats, Golden hamsters and guinea pigs were inoculated with 10 3.5 TCID 50 of VSV-LASV by the standardized intraperitoneal route. All animals were bred in BSL-2 laboratory conditions and were closely monitored daily for survival and specific neurological symptoms including tremor, ataxia, limb paralysis, convulsions, as well as activity, piloerection and suckling.
[0059] Results: All 3-day-old BALB / c suckling mice in the VSV-LASV IV Josiah infection group died, while the rest of the groups did not (as shown in Figure 1). This indicates that the VSV-LASV recombinant virus is not lethal to adult rodents but is highly lethal to suckling mice. Figure 10
[0060] In summary, the present application successfully constructed a recombinant chimeric virus (VSV-LASV) expressing the glycoprotein (GP) of the Lassa virus (LASV) Josiah strain based on the reverse genetics system of vesicular stomatitis virus (VSV). The recombinant virus induced lethal infection in 3-day-old BALB / c suckling mice and exhibited age and host species-dependent susceptibility characteristics: after standardized intraperitoneal infection (dose: 10 3.5 TCID 50 ), 3-day-old suckling mice exhibited progressive motor disorders, sharp weight loss and other neurological dysfunction symptoms at the late stage of infection (≥4 dpi) and all died within 6-11 dpi; dynamic analysis of viral load confirmed that it exhibited explosive replication in the central nervous system (CNS) Figure 6 ), this pathological process precisely mimics the rare viral neuroinvasion and concurrent encephalitis complications in human severe Lassa fever cases. Importantly, 5-day-old pups survived infection with 100% survival rate and no clinical symptoms, which is consistent with the age-dependent susceptibility trend observed in primate models, possibly due to the developmental defects of blood brain barrier (BBB) and immature immune responses in neonatal pups. This study not only confirms that LASV GP is a key virulence factor, but more importantly, reveals that its virulence is highly dependent on the genetic lineage of the virus, with the GP of IV-type Josiah strain exhibiting unique and strong pathogenicity in this model, and also found that it does not induce lethal infection in adult mice, golden hamsters, guinea pigs and rats, and other rodent models, showing good safety.
[0061] Based on the above characteristics, the present application has broken through to establish a Lassa fever alternative animal model that can be safely operated in a biosafety level two (BSL-2) laboratory. Compared with the wild-type LASV infection model which must rely on BSL-4 facilities, this system, through the specific neural tropism mediated by the GP protein, highly reproduces the core pathological link of LASV infection - cross-barrier transmission and neural pathogenesis, while avoiding the highest biosafety risk. This model not only provides a unique research tool for analyzing the neural invasion of LASV glycoprotein, but also significantly reduces the technical threshold and operating cost of Lassa virus neutralizing antibody evaluation, antiviral drug (especially drugs targeting neural complications or needing to penetrate BBB) high-throughput screening, and emergency vaccine potency verification, and has irreplaceable application value for promoting the development of high pathogenic virus prevention and control strategies.
[0062] The above-described embodiments are merely specific implementations of the present application, which are used to illustrate the technical solutions of the present application, but not to limit the same. The protection scope of the present application is not limited thereto, although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that any person skilled in the art can modify or easily think of changes to the technical solutions recorded in the foregoing embodiments, or make equivalent replacements to part of the technical solutions; and these modifications, changes or replacements do not make the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application. All should be covered within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.
Claims
1. A method for establishing a Lassa fever alternative suckling mouse infection model, characterized in that: Using the recombinant chimeric virus VSV-LASV at 10 3.5 TCID 50 The dose was administered via intraperitoneal injection to infect 3-day-old BALB / c suckling mice; the recombinant chimeric virus VSV-LASV was constructed using reverse genetics technology as a vesicular stomatitis virus (VSV) vector, in which the envelope glycoprotein GP gene in its genome was completely replaced by the Lassa virus (LASV) Josiah strain glycoprotein (GP) gene, thereby chimeric expression of LASV GP protein.
2. The method for establishing a Lassa fever alternative suckling mouse infection model as described in claim 1, characterized in that: The method for constructing the recombinant chimeric virus VSV-LASV includes: replacing the GP gene in the full-length plasmid of a VSV infectious clone carrying a GFP reporter gene tag with the GP gene of the LASV Josiah strain to construct the recombinant full-length plasmid pVSV-ΔG-GFP-LASV GP; co-transfecting the recombinant full-length plasmid with four helper plasmids expressing VSV nucleocapsid protein, pVSV-N, pVSVS-P, pVSVS-L and pVSV-G, into BSR cells; rescuing and amplifying the recombinant chimeric virus VSV-LASV carrying a GFP tag or a Luc tag and expressing LASV GP.
3. A Lassa fever alternative suckling mouse infection model, characterized in that, It is constructed using the method described in claim 1 or 2.
4. The application of the Lassa fever alternative suckling mouse infection model as described in claim 3 in experimental environments with biosafety level 2 or lower.
5. The application as described in claim 4, characterized in that: This includes research on the neuroinvasion and pathogenesis mechanisms mediated by Lassa virus glycoproteins; and evaluation of the in vivo protective effects of Lassa virus neutralizing antibodies, antiviral drugs, or vaccines.
6. The application as described in claim 5, characterized in that: The study on the mechanisms of nerve invasion and pathogenesis includes: elucidating the molecular pathways by which the virus crosses the blood-brain barrier after intraperitoneal infection; evaluating the delivery efficiency of antiviral drugs to the central nervous system, and verifying their effectiveness by significantly reducing viral load or alleviating neurological symptoms in infected suckling rat brain tissue.