Chikungunya virus attenuated by expressing nanoluciferase gene and application

Abstract

This invention discloses an attenuated chikungunya virus expressing a nanoluciferase gene, the nucleotide sequence of which is shown in SEQ ID NO:1. It is constructed by attenuating the virulence of the chikungunya virus LR2006-OPY1 strain by deleting 61 amino acids from positions 323 to 383 of the nsP3 protein and inserting a nanoluciferase gene after position 490 of the nsP3 protein. This virus model maintains similar growth characteristics to the wild-type virus but with significantly improved safety. Viral replication can be monitored in real time by detecting luciferase activity, making it suitable for drug screening at both cellular and animal levels. This provides an efficient and safe technical platform for the development of antiviral drugs against chikungunya virus.

Description

Technical Field

[0001] This invention belongs to the field of virus technology, specifically relating to an attenuated chikungunya virus expressing a nanoluciferase gene and its applications. Background Technology

[0002] Chikungunya virus (CHIKV) is primarily transmitted by Aedes albopictus and Aedes aegypti mosquitoes and has spread rapidly worldwide in recent years. Current research indicates that CHIKV poses a persistent threat to global public health. Infection with CHIKV can cause Chikungunya fever (CHIKF), whose main clinical manifestations are fever, rash, and significant joint pain, with joint symptoms lasting for months or even years. Some severe cases may also develop neurological complications and even lead to death. Although CHIKV has spread widely globally, there are currently no specific antiviral drugs targeting this virus. At present, clinical treatment for Chikungunya fever mainly focuses on symptomatic and supportive care, such as using nonsteroidal anti-inflammatory drugs (NSAIDs) to relieve symptoms like fever and joint pain. However, these treatments only alleviate clinical manifestations and cannot effectively inhibit viral replication or clear the viral infection, thus limiting their effectiveness. To further investigate the pathogenicity of CHIKV and facilitate the screening of antiviral drugs, there is an urgent need for tools that can track viral replication and transmission in real time in animal models. Meanwhile, due to the high infectivity of the CHIKV reportable virus, research and development related to CHIKV requires the use of biosafety level 3 (BSL-3) laboratory conditions, which greatly limits the research and application of CHIKV.

[0003] VLA1553 (IXCHIQ®) is the first live attenuated CHIKV vaccine to receive full approval from the U.S. Food and Drug Administration. It is derived from the ECSA genotype La Reunion strain (LR2006-OPY1), which contains a 183-amino acid deletion in the nsP3 gene. This deletion has been shown to inhibit viral replication and reduce virulence while maintaining growth and immunogenicity in a laboratory setting. However, the development process for this type of antiviral drug is extremely complex, resulting in long development cycles and high costs. Therefore, constructing an efficient, stable, and suitable technology platform for CHIKV antiviral drug screening is of significant research and application value. Summary of the Invention

[0004] To address the current difficulties in screening antiviral drugs against CHIKV, this invention provides an attenuated Chikungunya virus expressing a nanofluorescent enzyme gene and its application in screening anti-Chikungunya virus drugs.

[0005] The attenuated chikungunya virus LR2006-ΔnsP3-Nluc expressing the nanoluciferase gene of this invention is constructed by deleting 61 amino acids from positions 323 to 383 of the nsP3 protein and inserting the nanoluciferase gene (Nluc) after amino acid position 490 of the nsP3 protein, based on the chikungunya virus LR2006-OPY1 strain. Its nucleotide sequence is shown in SEQ ID NO:1.

[0006] The method for constructing an attenuated chikungunya virus expressing the nanoluciferase gene in this invention is as follows: (1) Construct the recombinant plasmid pACYC-LR2006-ΔnsP3-Nluc; (2) After linearizing the recombinant plasmid, RNA was obtained by in vitro transcription; (3) Transfect the RNA into the host cells to obtain transfected host cells; (4) Culture the transfected host cells to obtain attenuated chikungunya virus expressing the nanoluciferase gene.

[0007] This invention applies the attenuated chikungunya virus expressing the nanoluciferase gene to the screening of anti-chikungunya virus drugs.

[0008] The beneficial effects of this invention are: (1) The LR2006-ΔnsP3-Nluc virus provided by this invention is based on the high sensitivity and simplicity of nano-luciferase detection, and only a small amount of virus is needed to complete the rapid detection. In this invention, the deletion of nsP3 protein attenuates the virus and reduces the biosafety risk. The inserted Nluc gene can reflect the virus replication status in real time, which effectively improves the sensitivity and specificity of detection. This invention is a one-step detection method and does not require complicated operation steps, which greatly improves the speed and simplicity of detection. (2) The chikungunya virus drug screening method provided by this invention has extremely high sensitivity, strong specificity, and is quick and easy to operate, enabling efficient screening of anti-chikungunya virus drugs; the experimental results described in the examples show that LR2006-ΔnsP3-Nluc virus can accurately reflect the inhibitory effect of drugs on viral replication at the cellular level, EC 50 The test results are reliable; in animal models, in vivo imaging technology can monitor the dynamic distribution and replication of the virus in the body in real time, providing an intuitive technical means for evaluating drug efficacy. The virus model constructed in this invention has the same growth characteristics as the wild-type virus, but is safer and suitable for general biosafety laboratory operations, providing an important technical platform for drug development and prevention and control of this virus. Attached Figure Description

[0009] Figure 1 The growth curves of LR2006-ΔnsP3 and LR2006-ΔnsP3-Nluc cDNA (Figure A) and the luciferase expression results (Figure B) are shown. Figure 2 EC50 curve of 4'-FlU in U2OS cells LR2006-ΔnsP3-Nluc; Figure 3 Photographs of paw pad swelling in mice infected with LR2006-ΔnsP3-Nluc; Figure 4 The curve of paw pad swelling in mice infected with LR2006-ΔnsP3-Nluc; Figure 5 Results of viremia in mice infected with LR2006-ΔnsP3-Nluc; Figure 6 The curve showing the change in body weight in mice infected with LR2006-ΔnsP3-Nluc. Figure 7 The results show the survival rate of mice infected with LR2006-ΔnsP3-Nluc. Figure 8 The abdominal images of mice infected with LR2006-ΔnsP3-Nluc are in vivo imaging results. Figure 9 Quantitative analysis of abdominal fluorescence activity in mice infected with LR2006-ΔnsP3-Nluc via in vivo imaging. Figure 10 The image shows the back of a mouse after LR2006-ΔnsP3-Nluc infection; Figure 11 Quantitative analysis of dorsal fluorescence activity in mice infected with LR2006-ΔnsP3-Nluc via in vivo imaging. Figure 12 These are in vitro imaging results of tissues from mice infected with LR2006-ΔnsP3-Nluc. Figure 13 Quantitative analysis of in vitro fluorescence activity of LR2006-ΔnsP3-Nluc-infected mouse tissues. Detailed Implementation

[0010] The technical solution of the present invention will be further described in detail below through embodiments, but the content of the present invention is not limited thereto. Unless otherwise specified, the methods in this embodiment are conventional methods, and the materials and reagents used are obtained from commercial sources or prepared according to conventional methods unless otherwise specified. Example 1: Rescue of the truncated nsP3 LR2006 strain (LR2006-ΔnsP3) and the truncated nsP3 LR2006 strain carrying the reporter gene (LR2006-ΔnsP3-Nluc) 1. Construction of the truncated nsP3 form of LR2006 strain (LR2006-ΔnsP3) Using the LR2006-OPY1 (DQ443544) strain as a template, PCR amplification was performed using the high-fidelity enzyme Phanta Flash Master Mix to obtain two target fragments: LR-nsp1-3 (see positions 1-5094 of SEQ ID NO:2) and LR-nsp4-sp (positions 5068-11732 of SEQ ID NO:2). A Pac I restriction site and a T7 promoter were added sequentially to the 5' end of LR-nsp1-3, and a Not I restriction site was added to the 3' end of LR-nsp4-sp. The amplification primer sequences are shown in Table 1; the amplification system is shown in Table 2; and the amplification conditions are shown in Table 3. Table 1 Primer Sequences

[0011] Table 2 Phanta Flash Master Mix PCR Reaction System ; Table 3 Phanta Flash Master Mix PCR Reaction Conditions ; Note: a: For target segments with a length not exceeding 10kb, it is recommended to set the extension time to 5sec / kb; when the target segment length exceeds 10kb, the extension time should be set to 10sec / kb.

[0012] After purification, the two fragments were inserted into the pACYC177 vector using a kit to obtain the target product pACYC-LR2006-ΔnsP3.

[0013] 2. Construction of the truncated nsP3 strain of LR2006 carrying the reporter gene Using plasmids pACYC-LR2006-ΔnsP3 and pNL1.1 as templates, three viral fragments were amplified: LR-Δnsp1-3 (see positions 1-5416 of SEQ ID NO:1), LR-nsp4-C (see positions 5905-8705 of SEQ ID NO:1), and LR-sp (see positions 8677-12245 of SEQ ID NO:1), along with an Nluc reporter gene fragment (see positions 5387-5935 of SEQ ID NO:1). A Pac I restriction site and a T7 promoter were sequentially added to the 5' end of LR-Δnsp1-3, and a Not I restriction site was added to the 3' end of LR-nsp4-C. The amplification primer sequences are shown in Table 4; the amplification system is shown in Table 2; and the amplification conditions are shown in Table 3. Table 4 Primer Sequences ; The underlined portion in the table specifically refers to the Nluc group sequence, while the other positions are linker regions.

[0014] After purification, the above fragment was inserted into the pACYC177 vector using a kit to obtain the target product pACYC-LR2006-ΔnsP3-Nluc.

[0015] 3. Plasmid PCR linearization Using pACYC-LR2006-ΔnsP3 and pACYC-LR2006-ΔnsP3-Nluc plasmids as templates, the full-length viral genome was amplified. After obtaining the product, it was purified to obtain the purified linearized plasmid. The amplification primer sequences are shown in Table 5; the amplification system is shown in Table 2; and the amplification conditions are shown in Table 3.

[0016] Table 5 Primer Sequences ; 4. In vitro transcription and RNA purification Using purified linearized plasmid DNA as a template, viral RNA was prepared by in vitro transcription using Invitrogen's mMESSAGE mMMACHINE kit (AM1344). The in vitro transcription reaction was carried out according to the following system based on the concentration of linearized plasmid DNA, following the instructions. The reaction system and reaction conditions are shown in Table 6.

[0017] Table 6 Reaction System and Reaction Conditions ; Note: All reagents must be removed from -20℃ and thawed at 4℃ before use. 2×NTP / CAP and Enzyme Mix should be placed on ice before use. Add reagents strictly according to the order listed in the table above. 10×Reaction Buffer must be fully brought to room temperature before addition. Gently tap with your finger to mix.

[0018] After the in vitro transcription reaction was completed, the RNA was purified using the lithium chloride precipitation method according to the kit instructions. The specific procedure is as follows: (1) Add 1 μL of TURBO DNase and mix thoroughly. Incubate at 37°C for 15 min to remove DNA from the system. (2) Transfer all products to a 1.5 mL enzyme-free centrifuge tube, add 30 μL Nuclease-free Water and 30 μL LiCl to terminate the reaction; (3) After mixing thoroughly, freeze at -20℃ for at least 30 min; (4) Centrifuge at 4°C at maximum speed for 15 min to precipitate RNA; (5) Carefully remove the supernatant. Add 1 mL of 70% ethanol to wash the RNA and centrifuge at maximum speed for 10 min; (6) After removing the supernatant, let the precipitate stand at room temperature for 5 min to dry; (7) Add an appropriate amount of Nuclease-free Water to dissolve the RNA precipitate according to the amount of precipitate to obtain the target product RNA. Then detect the RNA concentration and purity, and store the target product RNA in a -80℃ freezer.

[0019] 5. RNA transfection was performed using DMRIE-C transfection reagent. No antibiotics were added during the Vero cell culture process prior to transfection. Vero cells were then evenly seeded into 12-well plates at a density of 1.5 × 10⁶ cells per well. 5 Cells were placed in a 37°C, 5% CO2 incubator. When the Vero cells reached 80% confluence, transfection was performed using DMRIE-C. The specific procedure is as follows: (1) Wash Vero cells twice with Opti-MEM medium, and do not discard the medium after the last wash; (2) Add 500 μL of Opti-MEM medium and 4 μL of DMRIE-C transfection reagent to a 1.5 mL enzyme-free centrifuge tube, and mix gently by shaking. (3) Add 3 μg of RNA to a centrifuge tube containing DMRIE-C and culture medium, and gently vortex to mix. (4) Discard the culture medium in the well and immediately add the complex to the washed cells; (5) Incubate at 37℃ in a 5% CO2 incubator for 5 hours; (6) After incubation, wash the cells once with 2% DMEM medium and replace with 2% DMEM medium; (7) Observe the cytopathic effect 24 hours after transfection. When the cytopathic effect exceeds 50%, collect the cell supernatant, which is the P0 generation virus.

[0020] 6. PO virus amplification culture and seed virus preparation The P0 virus was inoculated into BHK-21 cells for large-scale culture to prepare seed culture for subsequent experiments. The specific steps are as follows: (1) Seed BHK cells in T75 culture flasks. When the cell confluence reaches 90%, change the cell culture medium to 2% DMEM. (2) 200 μL of P0 virus was inoculated into T75 BHK-21 cells and cultured in a 37°C, 5% CO2 incubator; (3) After culturing for 48 hours, when more than half of the BHK-21 cells became diseased, the viral supernatant was collected, centrifuged at 4000 rpm for 10 min, aliquoted, clearly labeled and stored in a -80℃ freezer for use as viral seed liquid for subsequent experiments, thus obtaining LR2006-ΔnsP3 virus and LR2006-ΔnsP3-Nluc virus; (4) Perform plaque assays on the preserved virus seed solutions to determine the viral droplets.

[0021] Example 2: Identification of the LR2006-ΔnsP3-Nluc virus BHK-21 cells were evenly seeded into 12-well plates, with 1.5 × 10⁶ cells per well. 5 Cells were placed in an incubator at 37°C and 5% carbon dioxide. Infection experiments were conducted when the cell confluence reached 90%. Cells were infected with LR2006-ΔnsP3 and LR2006-ΔnsP3-Nluc viruses obtained in Example 1 at an MOI of 0.01. After inoculation, the cells were incubated in an incubator for 1.5 h. After incubation, the viral supernatant was discarded, and the cells were washed three times with PBS. Then, 2% DMEM medium was added, which was considered the end of the infection experiment.

[0022] Cell supernatant was collected and viral titer was measured at 0 h, 12 h, 24 h, 36 h, and 48 h after the infection experiment. The results are as follows: Figure 1 As shown in Figure A, the experiment showed that the amplification levels of LR2006-ΔnsP3 and LR2006-ΔnsP3-Nluc were consistent, indicating that the addition of the Nluc gene did not affect the growth curve of LR2006-ΔnsP3.

[0023] Cell supernatants were collected at 12h, 24h, 36h, and 48h post-infection, and the viral expression of Nlucose was detected using the Nano-Glo luciferase Assay System (Promega) kit. Fluorescence intensity (luciferase activity) indicates the Nlucose expression capacity. Results are shown below. Figure 1 As shown in Figure B, Nluc in LR2006-ΔnsP3-Nluc is normally expressed, and the expression level of Nluc increases with the duration of infection, proving that the experimentally obtained LR2006-ΔnsP3-Nluc can be rapidly detected at the cellular level. Furthermore, the viral infection status can be traced based on the stably expressed Nluc gene.

[0024] Example 3: Application of LR2006-ΔnsP3-Nluc virus in in vitro cell drug screening U2OS cells were seeded in 12-well plates at a density of 2 × 10⁶ cells per well. 5 Cells were placed in an incubator at 37°C and 5% carbon dioxide. When the cell confluence reached 90%, the experiment was conducted. The LR2006-ΔnsP3-Nluc virus obtained in Example 1 was used to prepare a virus dilution at MOI=0.1.

[0025] Viral inoculum was prepared by diluting 4'-fluorouridine (4'-FIU) with viral diluent to a final concentration of 10 μM. The drug was serially diluted in a 0.5 log10 gradient to obtain different concentrations of viral inoculum. Sterile water was used as a control. After adsorption in a 37°C, 5% CO2 incubator for one hour, the viral inoculum was removed and replaced with fresh, intact culture medium containing the same concentration of 4'-FIU. Cells were incubated at 37°C, 5% CO2 for 24 hours, and the cell culture supernatant was collected. Nluc activity was detected using the Nano-Glo luciferase Assay System (Promega) kit, and luciferase signals were collected using a microplate reader. The infection level of the control group was set at 100%, and the infection level of the drug treatment groups was normalized to this control group. The inhibition rate was calculated by subtracting the relative infection level of each treatment group from 100%. EC analysis was performed using GraphPad Prism software. 50 value.

[0026] like Figure 2 As shown, the inhibitory effect of the drug can be reflected by detecting the luciferase activity in the cell culture supernatant after drug addition, demonstrating that LR2006-ΔnsP3-Nluc can rapidly detect the EC50 of the drug at the cellular level.50 .

[0027] Example 4: Application of LR2006-ΔnsP3-Nluc virus in in vivo imaging of IFNAR1-deficient A6 mice All animal experiments used male and female mice aged 6-8 weeks, which underwent hair removal treatment three days before infection.

[0028] 1. The grouping is as follows: In all cases below, groups (1), (2), and (3) used DMEM basal medium to dilute the LR2006-ΔnsP3-Nluc virus obtained in Example 1, with a final inoculation dose of 10. 4 PFU was administered to mice via a combination of intradermal and footpad injections, while the blank control group received the same volume of DMEM basal medium. Specific groupings are as follows: (1) Vector treatment group: a total of 6 animals were treated with vector (10% DMSO, 5% Tween-80, 40% PEG300 and 45% saline) after viral inoculation; (2) 5 mg / kg / day treatment group: a total of 6 animals were treated with 5 mg / kg / day of 4′-FIU after viral inoculation; (3) 10 mg / kg / day treatment group: a total of 6 animals were treated with 4′-FIU at 10 mg / kg / day after viral inoculation; (4) Blank control group (MOCK): 3 mice were injected with the same volume of DMEM basal medium; All treatment groups were treated by gavage within 2 hours of infection, with the drugs dissolved in carriers (10% DMSO, 5% Tween-80, 40% PEG300 and 45% saline).

[0029] 2. Testing blood samples Following infection, 50 μL of whole blood samples were collected from mice via tail-tip sampling at the designed time points for viremia detection. Mice were treated once daily until the end of the experiment. To monitor disease progression, observations were made daily, including survival rate, weight change, severity of footpad swelling, and clinical symptoms.

[0030] (1) Detect the degree of swelling of the foot pads like Figure 3 , 4As shown, footpad swelling in the carrier-treated group developed rapidly post-infection, peaking on day 3. In the group treated with 5 mg / kg / day 4'-FlU, peak swelling was delayed until day 5, while the 10 mg / kg / day 4'-FlU group showed a further delay, with peak swelling occurring on day 9. These results indicate that 4'-FlU significantly inhibited the progression of footpad swelling in mice.

[0031] (2) Detection of viremia like Figure 5 As shown, viremia testing revealed that, compared with the vector control group, the viral load in the blood of all 4'-FlU treatment groups was significantly reduced.

[0032] (3) Detect weight and survival rate Regarding weight and survival rate, such as Figure 6 , Figure 7 As shown, mice in both the vector treatment group and those treated with 5 mg / kg / day 4'-FlU exhibited significant weight loss starting from day 2 post-infection. All mice in the vector control group died on day 3 post-infection. Mice treated with 5 mg / kg / day 4'-FlU began to recover weight from day 5, but all died within 7 days. Mice in the 10 mg / kg / day 4'-FlU group maintained stable weight and survived throughout the study.

[0033] 3. Detection of bioimaging Mice were anesthetized with 1.25% 2,2,2-tribromoethanol solution. Nano Glo® Fluorofurimazine (Promega) was prepared according to the manufacturer's instructions and then diluted with PBS to a 1:5 ratio before use. 100 μL of the diluted substrate was injected intraperitoneally into each mouse. Luciferase signals were collected using the IVIS Lumina LT (PerkinElmer) imaging system, a dedicated instrument designed for this type of application. Images of live animals were acquired under standardized exposure conditions with an exposure time of 40 seconds. All images were analyzed using Living Image software version 4.4 developed by Caliper Life Sciences. Quantification of bioluminescence signals was achieved by delineating regions of interest (ROIs) on uniformly scaled images. Data are expressed as total photon flux, defined as photons per second (p / s).

[0034] The results are as follows Figures 8-11As shown, the results of bioluminescence imaging further confirmed that the bioluminescence signal was significantly weakened in all 4'-FlU treatment groups compared to the carrier control group. Mice in the 10 mg / kg / day 4'-FlU group showed only weak signals in the abdominal and back regions. Quantitative analysis of the photon flux of the bioluminescence signal in the abdominal and back regions of mice also confirmed these observations, indicating that the signal intensity in the 10 mg / kg / day 4'-FlU group was significantly lower than that in the other two groups.

[0035] 4. Detect target organs Target organs were collected from each group of mice for further analysis. For in vivo imaging analysis, tissues were collected at 1 dpi, 2 dpi, and 3 dpi. Collected tissues included the brain, heart, spleen, kidneys, liver, and bilateral muscles. Following tissue collection, the excised organs were subjected to in vitro bioluminescence imaging with an exposure time of 60 seconds.

[0036] The results are as follows Figures 12-13 As shown, the bioluminescent signal intensity was significantly reduced in the 4'-FlU treatment group compared to the carrier control group. In mice treated with 5 mg / kg / day of 4'-FlU orally, weak signals were detected only in the kidneys and left leg muscles.

[0037] 5. Conclusion Based on the above experimental results, it can be seen that the LR2006-ΔnsP3-Nluc virus rescued by the LR2006-ΔnsP3-Nluc infectious clone constructed in this invention can be dynamically observed in mice, and the development process of the virus in the animal body can be observed. It can also be observed that the target organs in the mouse have the same bioluminescent signal trend, which has broad application value.

[0038] When treated with 4'-FlU at 5 mg / kg / day or 10 mg / kg / day, the luciferase activity in mice treated with the drug was significantly lower than that in the control group from day 1 to day 3. This indicates that the drug has a significant inhibitory effect on viral load compared with the control group. The LR2006-ΔnsP3-Nluc virus model can be used as an effective model for drug screening and evaluation in mice through in vivo imaging.

Claims

1. An attenuated chikungunya virus expressing a nanoluciferase gene, characterized in that: The nucleotide sequence is shown in SEQ ID NO:

1.

2. The use of the attenuated chikungunya virus expressing the nanoluciferase gene as described in claim 1 in screening anti-chikungunya virus drugs.

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