A recombinant chikungunya virus and uses thereof

Abstract

The present application relates to the technical field of biopharmaceutical medicine, and particularly relates to a recombinant chikungunya reporter virus and application thereof. The recombinant chikungunya reporter virus comprises: a recombinant virus nucleic acid, wherein the recombinant virus nucleic acid comprises: a genomic sequence of chikungunya virus, and a nucleotide sequence of an exogenous reporter gene; and the nucleotide sequence of the exogenous reporter gene is inserted into the C terminal of the nucleotide sequence encoding NS4 protein in the genomic sequence of the chikungunya virus. The present application obtains a new reporter virus by inserting the exogenous reporter gene into a specific position in the genomic sequence of the chikungunya virus. The reporter virus can successfully infect host cells, and is stably and continuously passed and expressed, and can be used as a molecular model for detecting virus replication. The reporter virus provided by the present application can also be used for screening of anti-chikungunya virus drugs and detection of neutralizing antibodies, and has important application value.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, and in particular to a recombinant chikungunya reporter virus and its applications. Background Technology

[0002] Chikungunya virus (CHIKV) is the pathogen that causes Chikungunya fever. It belongs to the genus *Alphavirus* of the family Togaviridae, and its genome is a single-stranded positive-sense RNA. CHIKV is primarily transmitted through the bite of Aedes mosquitoes, with *Aedes aegypti* and *Aedes albopictus* being the main vectors. Human infection with the virus causes Chikungunya fever, with an incubation period typically of 3–12 days. The main clinical manifestations include fever, fatigue, rash, headache, and joint pain. Some infected individuals may suffer from chronic joint pain for several years after the acute phase. Recent studies indicate that CHIKV infection may lead to more serious clinical consequences, including encephalitis and hemorrhagic manifestations. Currently, CHIKV is widespread globally, affecting more than 100 countries and regions, threatening not only individual health but also posing a serious global public health challenge. To date, there is no specific treatment for CHIKV infection, and vaccine development is still underway.

[0003] The CHIKV genome is approximately 11,800 nt in length and contains two open reading frames (ORFs). The first ORF encodes four non-structural proteins (nsP1~nsP4), which are mainly responsible for viral RNA synthesis and polymer processing. The second ORF encodes five structural proteins (C, E3-E2, 6K, E1), which are responsible for viral particle assembly and formation.

[0004] Reporter genes are crucial tools in modern molecular biology research for analyzing gene expression regulation, widely used in promoter analysis, signal transduction studies, receptor function identification, and drug screening. Enhanced Green Fluorescent Protein (EGFP) is a modified version of wild-type green fluorescent protein (GFP), significantly increasing its fluorescence intensity by replacing phenylalanine at position 64 with leucine. EGFP has a maximum excitation wavelength of 488 nm and a maximum emission wavelength of 510 nm. Due to its high sensitivity and ease of detection, the EGFP reporter gene system has become a universal technique in biological imaging, molecular biology, and biomedical research. Summary of the Invention

[0005] To address the technical problems existing in the prior art, this invention provides a recombinant chikungunya reporter virus and its applications.

[0006] In a first aspect, the present invention provides a recombinant viral nucleic acid, comprising: the genome sequence of Chikungunya virus, and the nucleotide sequence of an exogenous reporter gene; The nucleotide sequence of the exogenous reporter gene is inserted into the 3' end of the nucleotide sequence encoding the NS4 protein in the genome sequence of the chikungunya virus.

[0007] In the field of recombinant reporter virus construction, the insertion of large exogenous fragments (such as EGFP) often causes steric hindrance, leading to a significant decrease in viral replication efficiency or genomic instability (i.e., loss of the reporter gene during passaging). Especially at the junction of the nsp4 and C proteins, where the transcriptional fine-tuning of viral subgenomics and the assembly of structural proteins are involved, the insertion of exogenous sequences is likely to interfere with the normal life cycle of the virus.

[0008] However, during the research process, this invention discovered that precisely inserting EGFP between the nsp4 and C proteins not only did not cause the expected decrease in viral titer or packaging defects, but its unique spatial conformation also protected the EGFP sequence from viral genome recombination and knockout, thus exhibiting extremely high genetic stability after multiple passages. At the same time, in conjunction with the unique transcriptional kinetics of this region, the fluorescence signal intensity is also more stable, with lower background noise, combining high stability and fluorescence characteristics.

[0009] This invention is still under investigation, with the possibility of inserting the EGFP reporter gene at more common locations such as between nsp3 and nsp4, between nsp4 and C protein, and between E1 and 3'UTR. However, due to limitations such as steric hindrance or susceptibility to cleavage, it suffers from drawbacks such as limited fluorescence expression efficiency (instability leading to poor correlation between fluorescence intensity and viral titer) and long expression time.

[0010] Furthermore, the genome sequence of the chikungunya virus includes any of the following nucleotide sequences: (1) The nucleotide sequence as shown in SEQ ID NO.1; (2) The nucleotide sequence complementary to (1); (3) A nucleotide sequence that is at least 70, 80, 90 or 95% identical to the nucleotide sequence shown in (1) or (2).

[0011] The nucleotide sequence shown in SEQ ID NO.1:

[0012] The 3' end of the nucleotide sequence encoding the NS4 protein is the position following the 7565th bp of the nucleotide sequence shown in SEQ ID NO.1.

[0013] Furthermore, the exogenous reporter gene is the encoding gene for EGFP; Preferably, the coding gene for EGFP comprises any of the following nucleotide sequences: (1) The nucleotide sequence shown in SEQ ID NO.2; (2) The nucleotide sequence complementary to (1); (3) A nucleotide sequence that is at least 70, 80, 90 or 95% identical to the nucleotide sequence shown in (1) or (2).

[0014] The nucleotide sequence shown in SEQ ID NO.2: .

[0015] Secondly, the present invention provides a vector comprising: the aforementioned recombinant viral nucleic acid.

[0016] Preferably, the vector is an infectious cloning vector.

[0017] Thirdly, the present invention provides a recombinant chikungunya reporter virus, comprising the aforementioned recombinant viral nucleic acid, or obtained by rescuing the aforementioned vector in a host cell.

[0018] Fourthly, the present invention provides a host cell comprising: the aforementioned vector, or infected with the aforementioned recombinant chikungunya reporter virus.

[0019] Fifthly, the present invention provides a kit comprising the aforementioned recombinant viral nucleic acid, or the aforementioned vector, or the aforementioned recombinant chikungunya reporter virus, or the aforementioned host cell.

[0020] In a sixth aspect, the present invention provides a method for screening drugs against Chikungunya virus, comprising: infecting host cells with the aforementioned recombinant Chikungunya reporter virus; The host cells are cultured with or without the candidate drug; The anti-Chikungunya virus activity of the candidate drug is determined based on the changes in the expression level of the exogenous reporter gene in the recombinant Chikungunya reporter virus.

[0021] In a seventh aspect, the present invention provides a method for detecting neutralizing antibodies against chikungunya virus, comprising: mixing a serum to be tested with the aforementioned recombinant virus, and detecting the signal of an exogenous reporter gene in the recombinant chikungunya reporter virus.

[0022] Eighthly, the present invention provides the use of the aforementioned recombinant viral nucleic acid, or the aforementioned vector, or the aforementioned recombinant chikungunya reporter virus, or the aforementioned host cell in the preparation of a kit for screening anti-chikungunya virus drugs or detecting neutralizing antibodies against chikungunya virus.

[0023] The present invention has the following beneficial effects: This invention involves inserting a foreign reporter gene into a specific site on the chikungunya virus to obtain a novel reporter virus capable of infecting host cells and expressing viral proteins within the cells. Furthermore, the reporter virus provided by this invention can be continuously passaged and exhibits stable expression.

[0024] The report virus provided by this invention can be used for screening anti-Chikungunya virus drugs and evaluating the neutralizing titer of Chikungunya virus vaccine neutralizing antibodies, and has important application value in the biomedical field. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0026] Figure 1 This is the pSMART-CHIKV-GD133-EGFP genome splicing method provided in the embodiments of the present invention.

[0027] Figure 2This is a pSMART-CHIKV-GD133-EGFP plasmid map provided in the embodiments of the present invention; 26S is the non-coding region; 2SG is the subgenomic promoter.

[0028] Figure 3 This is the CHIKV gene expression structure in the infectious cloning plasmid of Chikungunya virus reporter virus (CHIKV-EGFP) provided in the embodiments of the present invention.

[0029] Figure 4 The graphs show the results of fluorescent protein expression at 12 / 24 / 36 / 48 hours for the P5 generation of Chikungunya virus reporter virus, as provided in this embodiment of the invention.

[0030] Figure 5 This is a graph showing the expression of the reporter gene 120 hours after HEK293T&Vero was transfected with the infectious clonal plasmid of the chikungunya virus reporter virus provided in this embodiment of the invention.

[0031] Figure 6 This invention provides an example of immunofluorescence detection of CHIKV envelope E1 protein in Vero cells.

[0032] Figure 7 This is a graph showing the fluorescence expression results of the reporter virus at different dilutions obtained from the embodiment of the present invention.

[0033] Figure 8 This is a graph showing the results of the constructed report virus detection neutralizing antibody provided in an embodiment of the present invention. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0035] Unless otherwise specified, the experimental methods involved in the following embodiments are conventional methods in the art. For example, you can refer to the experimental manual in the art or follow the conditions recommended in the manufacturer's instructions.

[0036] Unless otherwise specified, all experimental materials and reagents used in the following examples are commercially available.

[0037] Example 1: Construction method of Chikungunya virus reporter virus CHIKV-EGFP I. Chikungunya virus reporter virus genome segmentation and assembly (method as follows) Figure 1 (As shown).

[0038] Infective clones of chikungunya virus reporter virus (CHIKV-EGFP) were generated using pSMART-CHIKV-CMV-GD133 preserved in our laboratory as templates.

[0039] 1. Cloning of fragment A: Using pSMART-CHIKV-CMV-GD133 as a template and primers 1 and 2 as specific primers, the chikungunya virus fragment A was amplified by PCR.

[0040] Primer 1: 5'- CTTGACAATTAAGTATGAAGGTATATGTG-3'; Primer 2: 5'-CTCATAATAGACAACTTGCCTCGT-3'.

[0041] The PCR reaction system (100 μL) is as follows: 5×Q5 Reaction Buffer, 20μL; 10 mM dNTPs, 2 μL; 10 μM primers, 5 μL; 10 μM primers, 5 μL; Q5 High-Fidelity DNA Polymerase, 1μL; pSMART-CHIKV-CMV-GD133 cdna, 1μL; Nuclese-Free Water, 66 μL.

[0042] The PCR reaction conditions are as follows: Pre-denaturation at 98℃ for 30 seconds; One cycle consists of 98℃ for 10 seconds, 62℃ for 30 seconds, and 72℃ for 2 minutes; 35 cycles are performed. Finally, extend at 72°C for 2 minutes.

[0043] 2. Cloning of fragment B: Using pSMART-CHIKV-CMV-GD133 as a template and primers 3 and 4 as specific primers, the chikungunya virus fragment B was amplified by PCR.

[0044] Primer 3: 5'- GGCAAGTTGTCTATTATGAGAGGGAAAAAGCTAAAACCGT-3'; Primer 4: 5'- GAATCGGACGCTAGCCATGGGTGTTATATTCCCTTCTCTCTCGTC-3'.

[0045] The PCR reaction system (100 μL) is as follows: 5×Q5 Reaction Buffer, 20μL; 10 mM dNTPs, 2 μL; 10 μM primers, 5 μL; 10 μM primers, 5 μL; Q5 High-Fidelity DNA Polymerase, 1μL; pSMART-CHIKV-CMV-GD133 cdna, 1μL; Nuclese-Free Water, 66 μL; The PCR reaction conditions are as follows: Pre-denaturation at 98℃ for 30 seconds; One cycle consists of 98℃ for 10 seconds, 72℃ for 30 seconds, and 72℃ for 2 minutes and 45 seconds. 35 cycles are performed. Finally, extend at 72°C for 2 minutes.

[0046] 3. Cloning of the C fragment: Using pSMART-CHIKV-CMV-GD133 as a template and primers 5 and 6 as specific primers, the C fragment of Chikungunya virus was amplified by PCR.

[0047] Primer 5: 5'- CCATGGCTAGCGTCCGATTC-3'; Primer 6: 5'- tcctcgcccttgctcaccatCTATTTAGGACCGCCGTACA-3'.

[0048] The PCR reaction system (100 μL) is as follows: 5×Q5 Reaction Buffer, 20μL; 10 mM dNTPs, 2 μL; 10 μM primers, 5 μL; 10 μM primers, 5 μL; Q5 High-Fidelity DNA Polymerase, 1μL; pSMART-CHIKV-CMV-GD133 cdna, 1μL; Nuclese-Free Water, 66 μL.

[0049] The PCR reaction conditions are as follows: Pre-denaturation at 98℃ for 30 seconds; One cycle consists of 98℃ for 10 seconds, 72℃ for 30 seconds, and 72℃ for 2 minutes and 45 seconds. 35 cycles are performed. Finally, extend at 72°C for 2 minutes.

[0050] 4. Cloning of the D-EGFP fragment: Using pEGFP-N1 as a template, primers EGFP-F and EGFP-R as specific primers, the D-EGFP fragment was amplified by PCR.

[0051] Primer EGFP-F: 5'-atggtgagcaagggcgagga-3'; Primer EGFP-R: 5'- ttacttgtacagctcgtccatgc-3'.

[0052] The PCR reaction system is as follows: 5×Q5 Reaction Buffer, 20μL; 10 mM dNTPs, 2 μL; 10 μM primer EGFP-F, 5 μL; 10 μM primer EGFP-R, 5 μL; Q5 High-Fidelity DNA Polymerase, 1μL; pEGFP-N1 cdna, 1 μL; DMSO, 5 μL; Nuclese-Free Water, 61 μL.

[0053] The PCR reaction conditions are as follows: Pre-denaturation at 98℃ for 30 seconds; One cycle consists of 98℃ for 10 seconds, 69℃ for 30 seconds, and 72℃ for 30 seconds; 35 cycles are performed. Finally, extend at 72°C for 2 minutes.

[0054] 5. Cloning of the E fragment: Using pSMART-CHIKV-CMV-GD133 as a template and primers 7 and 8 as specific primers, the E fragment was amplified by PCR.

[0055] Primer 7: 5'- tggacgagctgtacaagtaaGTCATAACTTTGTACGGCGG-3'; Primer 8: 5'-CCTTCATACTTAATTGTCAAG-3'.

[0056] The PCR reaction system (100 μL) is as follows: 5×Q5 Reaction Buffer, 20μL; 10 mM dNTPs, 2 μL; 10 μM primers, 5 μL; 10 μM primers, 5 μL; Q5 High-Fidelity DNA Polymerase. 1μL; pSMART-CHIKV-CMV-GD133 cdna, 1μL; DMSO, 5 μL; Nuclese-Free Water, 61 μL.

[0057] The PCR reaction conditions are as follows: Pre-denaturation at 98℃ for 30 seconds; One cycle consists of 98℃ for 10 seconds, 56℃ for 30 seconds, and 72℃ for 2 minutes; 35 cycles are performed. Finally, extend at 72°C for 2 minutes.

[0058] 6. The five fragments A, B, C, D-EGFP and E obtained were subjected to agarose gel electrophoresis and gel extraction, including the following steps: Take 100 μL of PCR amplification product, add 10 μL of loading buffer, mix well and spot onto a 1% agarose gel, voltage 100V for 55 min; PCR product was extracted using a gel extraction kit, and the specific operation steps were performed according to the OmegaGel Extraction Kit D2500 kit instructions.

[0059] 7. The purified PCR product is recovered and purified using restriction endonucleases to cleave the G adenine methylation. m The ATC sequence digestion system was as follows: 1 μL DpnI, 2 μL 10×NEBuffer, ≤1 μg DNA, and deionized water to a final volume of 20 μL. The reaction mixture was incubated at 37°C for 40 min and then at 65°C for 20 min.

[0060] 8. The ligation system consisted of fragment A (77.7 ng), fragment B (92.4 ng), fragment C (86.36 ng), fragment D-EGFP (57.6 ng), fragment E (77.74 ng), 2×CloneExpress Mix (5 μL), and deionized water to a final volume of 10 μL. The reaction was carried out at 50 °C for 35 min, and then placed on ice after the reaction was completed.

[0061] 9. Add 7 μL of ligation product to 100 μL of EPI300 competent cells, mix well, and incubate on ice for 25 min. Heat shock at 42°C for 45 sec, then immediately place on ice for 2-3 min. Add 700 μL of antibiotic-free SOC medium, incubate at 37°C, shake at 200 rpm for 1 h, centrifuge at 5000 rpm for 1 min, discard 700 μL of supernatant, resuspend the remaining bacterial culture, plate it onto kanamycin-resistant LB medium, and incubate at 37°C for 12-16 h.

[0062] 10. Colony PCR: After the colonies have grown, pick a single colony and put it into 10 μL of deionized water. Primers 9 and 10 are specific primers, and colony PCR is performed.

[0063] Primer 9: 5'- AGTGCAGGGTATATCAGTTGTG-3'; Primer 10: 5'-aagcacgcgtaacctgttacc-3'.

[0064] The PCR reaction system (50 μL) is as follows: 5×Q5 Reaction Buffer, 10μL; 10 mM dNTPs, 1 μL; 10μM primer 9, 2.5μL; 10 μM primers, 10, 2.5 μL; Q5 High-Fidelity DNA Polymerase, 0.5μL; Bacterial suspension, 1 μL; Nuclease-Free Water, 32.5μL.

[0065] The PCR reaction conditions are as follows: Pre-denaturation at 98℃ for 30 seconds; One cycle consists of 98℃ for 10 seconds, 65℃ for 30 seconds, and 72℃ for 1 minute; 35 cycles are performed. Finally, extend the electrophoresis at 72℃ for 2 minutes; the bands of uniform size obtained after electrophoresis are the positive bacterial solutions.

[0066] 11. Add 10 μL of the single-clone positive bacterial culture to 15 mL of LB liquid medium containing kanamycin resistance, and incubate at 37°C in a shaker for 12-16 h. Send the bacterial culture for sequencing identification. For bacterial cultures with correct sequencing, extract the endotoxin-free plasmid, which is the EGFP reporter plasmid based on chikungunya virus-induced expression. Store at -20°C for later use. The endotoxin-free plasmid extraction procedure can be performed according to the instructions of the TIANGEN endotoxin-free plasmid mini-prep kit. The obtained plasmid is shown below. Figure 2As shown, the expression structure of the CHIKV gene in the infectious clone plasmid of Chikungunya virus reporter virus (CHIKV-EGFP) is as follows. Figure 3 As shown.

[0067] II. Transfection of infectious clonal plasmids of Chikungunya virus reporter virus (CHIKV-EGFP).

[0068] 1. Prepare Vero & HEK293T cells one day in advance. Prepare a cell suspension of Vero and HEK293T cells at a density of 1:1, seed them in a 6-well plate, and incubate overnight at 37°C in a 5% CO2 cell culture incubator.

[0069] 2. The next day, when the cell monolayer confluence reaches 80%-90%, the supernatant culture medium is discarded and replaced with new serum-free and antibiotic-free DMEM culture medium. The culture medium is then placed in a 37°C, 5% CO2 cell culture incubator for later use.

[0070] 3. Take 125 μL of Opti-MEM into an EP tube and dilute it thoroughly with 7.5 μL of Lipofectamine 3000 reagent.

[0071] 4. Take another 125 μL of Opti-MEM into an EP tube, add 2.5 μg of pSMART-CHIKV-EGFP plasmid to prepare a DNA premix, then add 5 μL of P3000 reagent (2 μL / μg DNA) and mix thoroughly.

[0072] 5. Mix the diluted DNA (step 3) with the diluted Lipofectamine 3000 reagent (step 4) at a 1:1 ratio and incubate at room temperature for 10 min.

[0073] 6. Add 250 μL of the DNA-liposome complex to each well of a 6-well plate, shake gently to mix, and incubate at 37°C in a 5% CO2 cell culture incubator. Replace the culture medium with DMEM containing 2% FBS as needed to monitor cell growth.

[0074] 7. After transfection, the fluorescence expression of enhanced green fluorescent protein (EGFP) was continuously observed using a fluorescence microscope.

[0075] 8. Harvest the supernatant after 120 hours and freeze it at -80℃ for future use as seed virus.

[0076] 120 hours after transfection, the expression of the reporter gene is as follows: Figure 4 As shown.

[0077] III. Stability of Chikungunya virus reportant virus.

[0078] 1. Prepare Vero cells in good condition one day in advance. Prepare a cell suspension of Vero cells, seed them in 6-well plates, and incubate overnight at 37°C in a 5% CO2 cell culture incubator. Once the cell density reaches a confluent monolayer, inoculate the reporter virus into the Vero cells and continuously observe the expression of enhanced green fluorescent protein (EGFP).

[0079] 2. Harvest virus particles P1 after 72 hours.

[0080] 3. After five consecutive passages, the report virus stably expresses enhanced green fluorescent protein (EGFP).

[0081] Chikungunya virus reporter virus was able to be continuously and stably passaged in Vero cells. The expression of fluorescent protein at P5 at 12 / 24 / 36 / 48 hours was as follows: Figure 5 As shown.

[0082] IV. Immunofluorescence detection of CHIKV envelope E1 protein in Vero cells.

[0083] 1. Prepare Vero cells in good condition one day in advance. Prepare a cell suspension of Vero cells, seed them in 6-well plates, and incubate overnight at 37°C in a 5% CO2 cell culture incubator. Once the cell density reaches a confluent monolayer, inoculate the Vero cells with reporter virus. After collecting the viral supernatant for 96 hours, wash the cell surface with PBS, digest the cells with 0.25% trypsin until the cells become like quicksand, then add 2 mL of 2% FBS DMEM medium to stop the digestion, prepare a cell suspension, and transfer it to a 1.5 mL Eppendorf tube.

[0084] 2. Centrifuge at 800 rpm for 5 minutes and discard the supernatant.

[0085] 3. Add, centrifuge at 800 rpm for 5 minutes, and discard the supernatant.

[0086] 4. Repeat the previous step twice.

[0087] 5. Add 400 μL of PBS to resuspend the cells, and take 20 μL of the cell suspension and drop it onto a glass slide.

[0088] 6. Dry under ultraviolet light for 2 hours.

[0089] 7. Transfer the slide to a staining jar containing a sufficient amount of pre-cooled acetone at 4°C and fix for 30 minutes.

[0090] 8. Wash the slide three times with PBS. After the slide surface is dry, add 20 μL of primary antibody (1% BSA to dilute the mouse ascites polyclonal antibody of CHIKV to 1:20) to the cells on the slide and incubate in a humidified chamber at 37°C for 30 min.

[0091] 10. Wash the slide three times with PBS. After the slide surface is dry, add 20 μL of secondary antibody (0.2% DAPI to dilute FITC-labeled goat anti-mouse IgG-Fc to 1:100) to the cells on the slide and incubate in a humidified chamber at 37°C in the dark for 30 min.

[0092] 11. Wash the slide three times with PBS, dry the surface of the slide, and observe the fluorescence distribution under a fluorescence microscope and take pictures.

[0093] 12. Observation under a fluorescence microscope revealed green fluorescence distribution in Vero cells infected with HEK293T & Vero cell culture medium transfected with the infectious clonal plasmid of Chikungunya virus reporter virus (CHIKV-EGFP) (FITC-labeled goat anti-mouse secondary antibody), while no green fluorescence was observed in the control group. Figure 6 This indicates that Vero cells infected with HEK293T & Vero cell culture medium transfected with the infectious clone plasmid of Chikungunya virus reporter virus (CHIKV-EGFP) can express the CHIKV envelope E1 protein.

[0094] Example 2 This embodiment further verifies the fluorescence expression of the reporter virus at different dilutions obtained in Example 1 to verify its stability, including the following procedures: 1. Prepare healthy Vero cells one day in advance. Prepare a cell suspension of Vero cells and seed them in 96-well plates for culture at 2×10⁶ cells / well. 3 / well, and incubate overnight in a 37°C, 5% CO2 cell culture incubator. Observe the cell status the next day and carry out subsequent experiments.

[0095] 2. Perform 10-fold serial dilutions of the reporter virus in EP tubes, discard the cell supernatant in the 96-well plate, wash twice with PBS, add 10-fold serial dilution of the reporter virus, set two replicates for each gradient, 200 μL of dilution in each well, discard the supernatant after 48 h, fix with 4% paraformaldehyde for 30 min, and take 3 readings with a microplate reader.

[0096] The results are as follows Figure 7 As shown, the reporter virus constructed in this invention exhibits high stability, high expression efficiency, and strong signal. It can capture an effective signal at a dilution of 1:1,000,000, and shows no significant fluorescence signal at a dilution of 1:10,000,000. It also shows no significant difference in OD value compared to the negative control. The reporter virus titer is 5 × 10⁻⁶. 6 The PFU / mL indicates that it has extremely high sensitivity.

[0097] Example 3 This embodiment further verifies the fluorescence stability of the reporter virus constructed in Example 1, including the following procedures: (1) Seed Vero cells in 96-well plates one day in advance, 2×10 3 cells / pores; (2) CHIKV-EGFP virus was administered at 200 PFU / 100 μL (4.0 × 10⁻⁶). 2 Neutralization assays were performed using an infectious dose of PFU, and the virus and serum antibodies were diluted using DMEM. The diluted virus solution (100 μL) and different dilutions of CHIKV murine ascites antibody (100 μL) were mixed in 96-well plates, and the virus-antibody mixture was incubated at 37°C for 1 hour. A negative antibody control and a virus-only infection control were also included, treated using the same methods.

[0098] (3) When the cells reach approximately 90% confluence, discard the old culture medium in the 96-well plate and add the pre-treated virus and antibody mixture (total volume 100 μL / well) to the cell surface. Set up two replicate wells for each antibody dilution. Incubate at 37°C, shaking the plate every 15 min. After 1 h, discard the virus and antibody mixture and wash once with sterile PBS. Then add 100 μL of 2% FBS DMEM cell culture medium and continue incubating at 37°C.

[0099] (4) After culturing for 48 hours, the fluorescence intensity was observed under a fluorescence microscope. The culture supernatant was discarded, and the cells were fixed with 4% paraformaldehyde. The EGFP fluorescence signal value was then detected using an enzyme-linked immunosorbent assay (ELISA) reader.

[0100] (5) Calculate the neutralizing activity of serum antibodies based on the ratio of the EGFP fluorescence signal value of the serum antibody-treated well to the fluorescence signal value of the virus-infected well without serum antibody treatment.

[0101] The results are as follows Figure 8 As shown, from Figure 8 The results show that the detection experiment has a high signal-to-noise ratio, and there is no specific interference in the control group. Furthermore, as the dilution factor of the murine ascites antibody increases (from 1:10 to 1:320), the relative infection rate of the virus shows a smooth, regular stepwise increase. This indicates that the intensity of the EGFP fluorescence signal can very accurately reflect the level of antibody neutralizing activity against the virus. The neutralizing ability gradually weakens with increasing ascites dilution, and the calculated neutralizing titer (NT50) is approximately 1:308.

[0102] The above results demonstrate that the CHIKV-EGFP reporter virus provided by this invention exhibits strong infectivity, stable fluorescence expression, and high sensitivity to neutralizing antibodies, making it suitable for the quantitative detection of CHIKV neutralizing antibodies. It also possesses the potential for efficient, high-throughput antibody screening or vaccine efficacy evaluation.

[0103] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A recombinant viral nucleic acid, characterized in that, include: The genome sequence of Chikungunya virus, and the nucleotide sequence of the exogenous reporter gene; The nucleotide sequence of the exogenous reporter gene is inserted into the 3' end of the nucleotide sequence encoding the NS4 protein in the genome sequence of the chikungunya virus.

2. The recombinant viral nucleic acid according to claim 1, characterized in that, The genome sequence of the chikungunya virus includes any of the following nucleotide sequences: (1) The nucleotide sequence as shown in SEQ ID NO.1; (2) The nucleotide sequence complementary to (1); (3) A nucleotide sequence that is at least 70, 80, 90 or 95% identical to the nucleotide sequence shown in (1) or (2).

3. The recombinant viral nucleic acid according to claim 1 or 2, characterized in that, The exogenous reporter gene is the encoding gene for EGFP; Preferably, the coding gene for EGFP comprises any of the following nucleotide sequences: (1) The nucleotide sequence shown in SEQ ID NO.2; (2) The nucleotide sequence complementary to (1); (3) A nucleotide sequence that is at least 70, 80, 90 or 95% identical to the nucleotide sequence shown in (1) or (2).

4. A carrier, characterized in that, include: The recombinant viral nucleic acid according to any one of claims 1-4.

5. A recombinant chikungunya reporter virus, characterized in that, It includes the recombinant viral nucleic acid as described in any one of claims 1-3, or obtained by rescuing the vector described in claim 4 from a host cell.

6. A host cell, characterized in that, Includes the vector of claim 4, or infected with the recombinant chikungunya reporter virus of claim 5.

7. A reagent kit, characterized in that, include: The recombinant viral nucleic acid according to any one of claims 1-3, or the vector according to claim 4, or the recombinant chikungunya reporter virus according to claim 5, or the host cell according to claim 6.

8. A method for screening drugs against Chikungunya virus, characterized in that, include: The recombinant chikungunya reporter virus described in claim 5 was used to infect host cells; The host cells are cultured with or without the candidate drug; The anti-Chikungunya virus activity of the candidate drug is determined based on the changes in the expression level of the exogenous reporter gene in the recombinant Chikungunya reporter virus.

9. A method for detecting neutralizing antibodies against Chikungunya virus, characterized in that, include: The serum to be tested was mixed with the recombinant chikungunya reporter virus of claim 5, and the signal of the exogenous reporter gene in the recombinant chikungunya reporter virus was detected.

10. The use of the recombinant viral nucleic acid of any one of claims 1-3, or the vector of claim 4, or the recombinant chikungunya reporter virus of claim 5, or the host cell of claim 6 in the preparation of a kit for screening anti-chikungunya virus drugs or detecting neutralizing antibodies against chikungunya virus.

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