Marburg hemorrhagic fever suckling mouse infection model capable of being operated in biosafety secondary laboratory and application of Marburg hemorrhagic fever suckling mouse infection model

Abstract

The invention discloses a Marburg hemorrhagic fever suckling mouse infection model capable of being operated in a biosafety secondary laboratory and application of the Marburg hemorrhagic fever suckling mouse infection model, and belongs to the technical field of biology. According to the invention, based on a VSV reverse genetics technology platform, a recombinant VSV-Angola expressing Marburg virus glycoprotein (GP) is constructed, the VSV-Angola is used as an alternative virus to cause lethal infection of 3-day-old BALB / c suckling rats, and a Marburg virus (MARV) alternative animal infection model adapted to biosafety secondary (BSL-2) laboratory operation is successfully developed. According to the infection model, the space load and the operation risk of a biosafety secondary laboratory are greatly reduced, a standardized neck subcutaneous inoculation technology is supported, the repeatability and the safety of an experiment are remarkably improved, the expenditure budget of an experimental animal is saved, and the infection model is suitable for research on a neuropathogenic mechanism and blood brain barrier penetrability of an antiviral drug; and the method has high cost benefit and clinical transformation value.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a Marburg hemorrhagic fever suckling mouse infection model that can be operated in a biosafety level 2 laboratory and an application thereof. Background Art

[0002] Marburg virus disease (MVD) is a severe zoonosis caused by Marburg virus (MARV). Marburg virus, a member of the genus Marburgvirus in the family Filoviridae, is a single-stranded, negative-sense RNA virus characterized by a typical filamentous structure. It can cause a systemic hemorrhagic fever syndrome, characterized by acute fever, coagulopathy, and multiple organ failure. Currently, the development of neutralizing antibodies against MARV and clinical trials of VSV-based vaccines are at a critical stage of advancement, but the virus's high mutation rate and immune escape characteristics still pose significant challenges to vaccine development.

[0003] The core challenge in studying MARV stems from its classification as a Biosafety Level 4 (BSL-4) pathogen. Experiments must be conducted in BSL-4 laboratories, requiring specialized laboratory conditions and sustained funding. Therefore, developing model virus systems (such as pseudovirus technology) and alternative animal infection models (such as golden hamster and hamster models) that can be safely operated in low-biosafety level laboratories has become a key path to breaking through research bottlenecks and accelerating the development of treatments and preventive measures.

[0004] The reverse genetics technology system based on Vesicular Stomatitis Virus (VSV) has become a core platform for constructing pathogen model viruses due to its high genomic plasticity, controllable biosafety risks (BSL-2 level), and efficient exogenous gene expression capabilities. By embedding the target virus surface glycoprotein (such as MARV-GP) into the VSV backbone, the cell invasion mechanism and neutralization epitope characteristics of the wild-type virus can be accurately simulated. It can not only achieve receptor binding and membrane fusion processes equivalent to those of the natural virus, but can also be used for high-throughput neutralizing antibody detection, vaccine immunogenicity assessment, and cross-species infection mechanism analysis. In the context of a lack of high-level biosafety laboratory resources, this system provides a key alternative strategy for basic research on BSL-4 pathogens such as Marburg virus. It can replace real viruses for antibody detection and establishment of animal infection models, and provides a scalable solution to accelerate antiviral drug screening and new vaccine development.

[0005] The present invention aims to construct an economical, efficient, and biosafety level 2 (BSL-2) laboratory-adaptable Marburg virus (MARV) alternative animal infection model based on the VSV reverse genetics technology platform. Summary of the Invention

[0006] One of the purposes of the present invention is to provide a Marburg hemorrhagic fever suckling mouse infection model that can be operated in a biosafety level 2 laboratory. The Marburg hemorrhagic fever suckling mouse infection model uses a recombinant VSV virus to infect 3-day-old BALB / c suckling mice. The recombinant VSV virus is a recombinant vesicular stomatitis virus in which the G gene of VSV is replaced with the GP gene of the Marburg virus Angola strain, referred to as VSV-Angola. The infection dose of the Marburg hemorrhagic fever suckling mouse infection model is 10 6.2 TCID 50 The route of infection is subcutaneous injection in the neck.

[0007] Furthermore, the VSV recombinant virus was constructed as follows: the G gene in the full-length plasmid of the VSV infectious clone carrying the eGFP tag was replaced with the GP gene of the Marburg virus Angola strain to obtain the MARV recombinant VSV full-length plasmid; the MARV recombinant VSV full-length plasmid was co-transfected with four VSV helper plasmids into BSR cells to rescue the MARV GP VSV carrying the eGFP tag, referred to as VSV-Angola.

[0008] The second object of the present invention is to provide a method for establishing the Marburg hemorrhagic fever suckling mouse infection model that can be operated in a biosafety level 2 laboratory. The recombinant VSV virus is used to infect 3-day-old BALB / c suckling mice at an infection dose of 10 6.2 TCID 50 The infection route is subcutaneous injection in the neck; the recombinant VSV virus is a recombinant vesicular stomatitis virus in which the G gene of VSV is replaced with the GP gene of the Marburg virus Angola strain, referred to as VSV-Angola.

[0009] A third object of the present invention is to provide the use of the above-mentioned Marburg hemorrhagic fever suckling mouse infection model that can be operated in a biosafety level 2 laboratory as an animal model for studying the attack mechanism of Marburg virus and evaluating the efficacy of Marburg virus antibodies or Marburg virus vaccines.

[0010] Furthermore, the application environment is a biosafety level 2 laboratory and below.

[0011] Furthermore, the study of the attack mechanism of Marburg virus is a study of the neuropathogenic mechanism and the blood-brain barrier penetration of antiviral drugs.

[0012] Compared with the prior art, the technical effects of the present invention are:

[0013] Based on the VSV reverse genetics technology platform, the present invention constructed a recombinant vesicular stomatitis virus (VSV-Angola) expressing Marburg virus glycoprotein (GP), and used it as a surrogate virus to cause lethal infection in 3-day-old BALB / c suckling mice. A Marburg virus (MARV) surrogate animal infection model suitable for biosafety level 2 (BSL-2) laboratory operations was successfully developed.

[0014] The present invention uses 3-day-old BALB / c suckling mice to construct a Marburg virus alternative infection model. Compared with the traditional golden hamster model, the 3-day-old BALB / c suckling mice weigh only 1.5-2.5g, and the experimental animal capacity per unit breeding area is increased by more than 30 times, which greatly reduces the space load and operational risks of the biosafety level 2 (BSL-2) laboratory; their small size characteristics support standardized neck subcutaneous inoculation technology, which controls the operational variation coefficient to below 5%, significantly improving the repeatability and safety of the experiment. At the same time, the cost per mouse is low, which can save more than 90% of the experimental animal funding budget in large-scale studies.

[0015] After infection with the VSV-MARV recombinant virus, the model showed specific infection characteristics in the immune-privileged areas (brain and eyes), accurately simulating the typical pathological process of the virus breaking through the blood-brain barrier and invading the central nervous system in human infection. Compared with the existing hamster model based on liver and spleen targeted infection, it is more suitable for the study of neuropathogenic mechanisms and blood-brain barrier penetration of antiviral drugs, and has both high cost-effectiveness and clinical translation value.

[0016] Furthermore, compared to the traditional golden hamster model, this suckling mouse model, combined with a small animal in vivo imaging system, can better demonstrate real-time in vivo viral tracking. This technological system overcomes the constraints of high-level biosafety laboratories, such as those required for highly pathogenic virus research, and provides standardized tools for studying cross-species pathogen transmission mechanisms and developing emergency vaccines and drugs. This is highly consistent with the strategic need for "scalable alternative models" advocated by the WHO's Priority Pathogen Research Roadmap. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments described in the present invention. For ordinary technicians in this field, other drawings can also be obtained based on these drawings.

[0018] Figure 1 This is a schematic diagram of the structure of the full-length recombinant VSV plasmid of the MARV Angola strain provided in an embodiment of the present invention.

[0019] Figure 2VSV-Angola recombinant pseudovirus rescue mode diagram provided in the embodiment of the present invention

[0020] Figure 3 These are the morphological observation results of the recombinant VSV virus provided in the embodiments of the present invention under a transmission electron microscope.

[0021] Figure 4 The weight changes and survival rates of BALB / c suckling mice of different ages infected with VSV-Angola provided in the embodiments of the present invention are shown.

[0022] Figure 5 The weight changes and survival rates of 3-day-old BALB / c suckling mice infected with VSV-Angola under different infection routes provided in the embodiments of the present invention.

[0023] Figure 6 LD of 3-day-old BALB / c suckling mice infected with VSV-Angola provided in the embodiment of the present invention 50 .

[0024] Figure 7 The viral load of VSV-Angola in the tissues of 3-day-old BALB / c suckling mice provided in the embodiments of the present invention is shown.

[0025] Figure 8 This is the dynamic monitoring of the viral tropism of VSV-Angola in 3-day-old BALB / c suckling mice provided by the embodiment of the present invention. DETAILED DESCRIPTION

[0026] To help those skilled in the art better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to the embodiments and accompanying drawings. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments derived by those skilled in the art based on the embodiments of the present invention without creative effort are also within the scope of protection of the present invention.

[0027] Unless otherwise specified, the test materials used in the following examples were purchased from conventional biochemical reagent stores. The experimental methods in the following examples were conventional methods unless otherwise specified.

[0028] The Marburg virus strain selected in the present invention is the Angola strain, and the VSV virus used is from a Military Veterinary Research Institute.

[0029] Example 1 Identification of MARV GP recombinant VSV

[0030] 1.1 Rescue and subculture of recombinant VSV virus

[0031] The present invention replaces the G gene in the full-length plasmid of VSV infectious clone carrying eGFP tag (p3.1-VSVΔG-eGFP-Angola) with the GP gene of Marburg virus (MARV) Angola strain by molecular cloning technology to construct the recombinant plasmid p3.1-VSVΔG-eGFP-Angola (such as Figure 1 Virus rescue was performed using a reverse genetics strategy: the recombinant full-length plasmid p3.1-VSVΔG-eGFP-Angola was co-transfected with four VSV helper plasmids p3.1-VSV-N, p3.1-VSV-P, p3.1-VSV-L, and p3.1-VSV-G (abbreviated as pN, pP, pL, and pG) in standard proportions into BSR cells (as shown in Figure 2 ). Figure 2 shown).

[0032] The specific procedure is as follows: BSR cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) until a monolayer density of 80% was achieved in a six-well plate. Transfection was then performed using the calcium phosphate method, following the specific steps in the kit instructions. Following transfection, the cells were incubated at 37°C and 5% CO₂ for 18 hours. Subsequently, the cells were shocked with 10% DMSO in PBS for 2.5 minutes. The supernatant was discarded and the cells were incubated in DMEM supplemented with 5% FBS for an additional 60 hours. Five passages were performed on Vero E6 cells, demonstrating that the recombinant virus could propagate and cause cytopathic effects in the cells.

[0033] Results showed that the harvested viral supernatant was inoculated into Vero E6 cells after three freeze-thaw cycles. After incubation at 37°C, 5% CO₂ for 1 hour, the cells were replaced with DMEM supplemented with 2% FBS for 48 hours. Stable cytopathic effect (CPE) was observed after five serial passages on Vero E6 cells, confirming that the recombinant virus (designated rVSVΔG-eGFP-AngolaGP, or VSV-Angola) was successfully rescued and replication-competent. The recombinant viral supernatant was aliquoted and stored at -80°C until further use.

[0034] 1.2 Electron microscopic observation of recombinant VSV virus

[0035] To evaluate the effect of MARV GP protein on the morphology of the recombinant virus, 100 μL of the supernatant of the 5th passage VSV-Angola virus was negatively stained with 2% phosphotungstic acid (pH 6.8) and then observed by transmission electron microscopy.

[0036] The results show (such as Figure 3 (As shown in Figure 2), both the recombinant and parental VSV viruses exhibited a typical bullet-shaped structure with evenly distributed fibrils on the surface. Electron microscopic analysis revealed that heterologous expression of the GP protein did not significantly alter the integrity of the viral capsid assembly, confirming that viral morphological characteristics were preserved.

[0037] Example 2 Establishment of VSV-Angola infection model

[0038] 2.1 Study on the lethality of VSV-Angola in BALB / c suckling mice

[0039] The present invention will replace the virus VSV-Angola with 10 6.2 3-day-old BALB / c suckling mice (referred to as P3) and 5-day-old BALB / c suckling mice (referred to as P5) were infected subcutaneously in the neck with a TCID50 dose. Control animals (3-day-old BALB / c suckling mice and 5-day-old BALB / c suckling mice) were injected subcutaneously in the neck with the same volume of PBS (referred to as Uninfected P3 and Uninfected P5). All animals were monitored daily for signs of disease, including changes in body weight, behavioral activity, and food and water intake.

[0040] The results showed that among the BALB / c suckling mice of different ages tested, VSV-Angola only caused lethal infection in 3-day-old BALB / c suckling mice. Figure 4 The lethal infection occurred in 5-day-old BALB / c suckling mice ( Figure 4 All of them survived and gained about 100% of their body weight (e.g. Figure 4 However, infected 3-day-old BALB / c suckling mice gained approximately 50% of their body weight at 4 dpi, but subsequently lost weight and all died within 5-7 dpi (as shown in FIG. Figure 4 In contrast, the control group of BALB / c suckling mice (Uninfected P3, Uninfected P5) survived and were euthanized as planned on day 15 after infection.

[0041] In addition, the present invention also analyzes the effects of two different infection routes of VSV-Angola, namely, subcutaneous injection (SC) in the neck and intraperitoneal injection (IP), on the lethality of 3-day-old BALB / c suckling mice. At the same time, two control groups (Uninfected SC and Uninfected IP) were injected with the same volume of PBS for subcutaneous injection and intraperitoneal injection in the neck; the characteristics of 3-day-old BALB / c suckling mice after infection with VSV-Angola were observed, and the LD of 3-day-old BALB / c suckling mice infected with VSV-Angola was determined. 50 ; Detect the viral load of the virus in various organs at different times after VSV-Angola infected 3-day-old BALB / c suckling mice; and detect the dynamic distribution of VSV-Angola in 3-day-old BALB / c suckling mice.

[0042] The results showed that 3-day-old BALB / c suckling mice infected by intraperitoneal injection (IP) lost weight and all died within 2-3 dpi. The lethal process was relatively fast and could not fully simulate the progressive pathological process of nerve damage in human MARV infection (e.g. Figure 5 3-day-old BALB / c suckling mice (SC) infected via subcutaneous injection in the neck gained approximately 50% of their body weight at 4 dpi, but subsequently lost weight and all died between 5 and 7 dpi. All BALB / c suckling mice in the control group (Uninfected SC, Uninfected IP) survived and gained weight.

[0043] 2.2 Characteristics of 3-day-old BALB / c suckling mice infected with VSV-Angola

[0044] In order to further study the characteristics of VSV-Angola infection in 3-day-old BALB / c suckling mice, the present invention designed five groups of 3-day-old BALB / c suckling mice, infected with different doses of VSV-Angola (10 2.2 TCID 50 , 10 3.2 TCID 50 , 10 4.2 TCID 50 , 10 5.2 TCID 50 , 10 6.2 TCID 50 ). Among them, 10 6.2 TCID 50 The mortality rate of infected suckling mice was 100%, 5.2 TCID 50 The mortality rate of infected suckling mice was 71%, 10 4.2 TCID 50 The mortality rate of infected suckling mice was 33%, 10 3.2 TCID 50 The mortality rate of infected suckling mice was 22%, 10 2.2 TCID 50 The mortality rate of infected suckling mice was 22%. At the same time, the infected suckling mice gained weight slowly and 6.2 TCID 50 The infected suckling mice began to lose weight at 4 dpi, while all the suckling mice in the uninfected control group survived and gained weight. Figure 6 shown.

[0045] To further investigate the replication of VSV-Angola in 3-day-old BALB / c suckling mice, the viral loads in tissues including liver, spleen, lung, kidney, brain, and eye were collected at 2, 4, 6, and 8 dpi and the TCID 50 The results showed that VSV-Angola could be detected in the spleen, lung, kidney, brain and eye tissues of 3-day-old BALB / c suckling mice infected with VSV-Angola, with the highest viral load in the brain (10 8.2 TCID 50 / g), followed by eyes (10 7.3 TCID 50 / g), and the viral load gradually increased with the course of the disease, and no virus was detected in the tissues of the uninfected control group (Uninfected). Figure 7 shown.

[0046] In addition, the present invention also conducted dynamic monitoring of the viral tropism of VSV-Angola in 3-day-old BALB / c suckling mice using an in vivo fluorescence imaging system to examine the tropism and distribution of VSV-Angola in 3-day-old BALB / c suckling mice at different times. Specifically, the viral signal intensity increased exponentially with the progression of the disease, and the virus gradually increased with the course of the disease, mainly present in the brain and central nervous system, such as Figure 8 Quantitative analysis showed that ( Figure 8 ), with fluorescence intensity peaking in the brain region at 6 dpi. This distribution pattern was consistent with the results of tissue viral load analysis, confirming that the recombinant virus successfully mimicked the neuroinvasive properties of the native virus.

[0047] In summary, the present invention constructed a recombinant vesicular stomatitis virus (VSV-Angola) expressing the Marburg virus glycoprotein (GP) through reverse genetics technology and used it as a surrogate virus to cause lethal infection in 3-day-old BALB / c suckling mice. After 3-day-old BALB / c suckling mice were infected subcutaneously with VSV-Angola in the neck, they began to show symptoms such as weight loss, movement disorders, visual impairment, and high viral loads in target organs at 4 days post-infection. All animals died within five to nine days after infection, and their clinical manifestations and pathological features were similar to the clinical course of human MVD. Notably, the lethality of this model showed significant age-dependence, with a 100% survival rate in 5-day-old suckling mice after infection, a phenomenon consistent with the age-related susceptibility trend observed in primates.

[0048] Therefore, the alternative virus infection model provided by the present invention that can be operated in a biosafety level 2 laboratory to simulate the symptoms of human Marburg virus disease breaks through the traditional BSL-4 restrictions and provides key technical support for the evaluation of the protective effect of MARV antibodies and the high-throughput screening of new antiviral drugs.

[0049] The above-described embodiments are only specific implementation methods of the present application, which are used to illustrate the technical solutions of the present application, rather than to limit them. The scope of protection of the present application is not limited thereto. Although the present application has been described in detail with reference to the above-mentioned embodiments, those skilled in the art should understand that any person skilled in the art can modify or easily conceive of changes to the technical solutions described in the above-mentioned embodiments within the technical scope disclosed in the present application, or perform equivalent replacements for some of the technical features thereof; and these modifications, changes or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present application. They should all be included in the scope of protection of the present application. Therefore, the scope of protection of the present application shall be subject to the scope of protection of the claims.

Claims

1. A Marburg hemorrhagic fever suckling mouse infection model that can be operated in a biosafety level 2 laboratory, characterized in that: The Marburg hemorrhagic fever suckling mouse infection model uses a recombinant VSV virus to infect 3-day-old BALB / c suckling mice. The recombinant VSV virus is a recombinant vesicular stomatitis virus in which the G gene of VSV is replaced with the GP gene of the Marburg virus Angola strain, referred to as VSV-Angola. The infection dose of the Marburg hemorrhagic fever suckling mouse infection model is 10 6.2 TCID 50 The route of infection is subcutaneous injection in the neck.

2. The Marburg hemorrhagic fever suckling mouse infection model operable in a biosafety level 2 laboratory according to claim 1, characterized in that: The VSV recombinant virus was constructed as follows: the G gene in the full-length plasmid of the VSV infectious clone carrying the eGFP tag was replaced with the GP gene of the Marburg virus Angola strain to obtain the MARV recombinant VSV full-length plasmid; the MARV recombinant VSV full-length plasmid was co-transfected with four VSV helper plasmids into BSR cells to rescue the MARV GPVSV carrying the eGFP tag, referred to as VSV-Angola.

3. The method for establishing a Marburg hemorrhagic fever suckling mouse infection model that can be operated in a biosafety level 2 laboratory according to claim 1, wherein: The recombinant VSV virus was used to infect 3-day-old BALB / c suckling mice at an infection dose of 10 6.2 TCID 50 The infection route is subcutaneous injection in the neck; the recombinant VSV virus is a recombinant vesicular stomatitis virus in which the G gene of VSV is replaced with the GP gene of the Marburg virus Angola strain, referred to as VSV-Angola.

4. The method for establishing a Marburg hemorrhagic fever suckling mouse infection model that can be operated in a biosafety level 2 laboratory according to claim 3, wherein: The VSV recombinant virus was constructed as follows: the G gene in the full-length plasmid of the VSV infectious clone carrying the eGFP tag was replaced with the GP gene of the Marburg virus Angola strain to obtain the MARV recombinant VSV full-length plasmid; the MARV recombinant VSV full-length plasmid was co-transfected with four VSV helper plasmids into BSR cells to rescue the MARV GP VSV carrying the eGFP tag, referred to as VSV-Angola.

5. Use of the Marburg hemorrhagic fever suckling mouse infection model operable in a biosafety level 2 laboratory as claimed in claim 1 as an animal model for studying the attack mechanism of Marburg virus and evaluating the efficacy of Marburg virus antibodies or Marburg virus vaccines.

6. The use according to claim 5, characterized in that The application environment is biosafety level 2 laboratory and below.

7. The use according to claim 5, characterized in that The Marburg virus attack mechanism study is a study of the neuropathogenic mechanism and the blood-brain barrier penetration of antiviral drugs.

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