Foot-and-mouth disease pseudovirus cell culture system and application thereof

Abstract

The invention discloses a foot-and-mouth disease pseudovirus cell culture system and application thereof. The invention belongs to the technical field of biology. The cell culture system provided by the invention can be used for producing foot-and-mouth disease pseudoviruses (FMDV-trVLPs) with transcription and replication capabilities, but does not have the infectivity of live viruses. The system is composed of a subgenome replicon (FMDV-Nluc) in which an FMDV capsid protein gene (P1) is replaced by Nluc and a BHK-21 cell line (BHK-P1) in which an FMDV P12A3C gene is stably integrated. When the FMDV-Nluc replicon is transfected into the BHK-P1 cell, the foot-and-mouth disease pseudovirus FMDV-trVLPs can be produced and can be proliferated and passaged in the BHK-P1 cell, so that biological safety degradation is effectively realized, and the FMDV-Nluc replicon has huge potential and practical value in the aspects of reducing biological safety risk and simulating FMDV live virus research.

Description

Technical Field

[0001] The present invention relates to a foot-and-mouth disease pseudovirus cell culture system and its application. The present invention belongs to the field of biotechnology. Background Art

[0002] Foot-and-mouth disease virus (FMDV) is a highly contagious and economically destructive small RNA virus. The virus particle is non-enveloped, has icosahedral symmetry, and encloses an 8.4 kb single-stranded positive-sense RNA genome. The FMDV genome contains a large open reading frame that encodes eight nonstructural proteins and four structural proteins: the nonstructural proteins include L pro , 2A, 2B, 2C, 3A, 3B, 3C and 3D, which mainly regulate the maturation and replication of foot-and-mouth disease virus; structural proteins VP1, VP2, VP3 combine to form the capsid surface structure, and VP4 forms the internal structure of the virus particle; structural protein VP1 is located on the surface of the virus and is a key capsid protein for virus adsorption, internalization, and replication. It plays an important role in the virus's fight against the host's immune system. It is an important antigenic site of FMDV and a key protein for FMDV mutation. In addition, FMDV can spread rapidly through direct or indirect routes. [6] , and its susceptible animals include more than 70 even-toed ungulates such as pigs, cattle, and sheep. Infected animals often show obvious clinical symptoms, such as fever, lameness, abnormal increase in salivation, and vesicular lesions on the hooves, which seriously affect the health and production performance of animals. In view of the serious impact of foot-and-mouth disease on the development of animal husbandry, the obstruction of international trade, and the potential threat to public health and safety, the World Organization for Animal Health (WOAH) has listed it as the first of the must-report animal diseases. my country has listed it as a Class I animal disease and implemented compulsory vaccination measures.

[0003] In view of the serious threat posed by FMDV to public health and the economy, the research on live FMDV virus must be carried out in biosafety level 3 laboratories (BSL-3) or above to ensure safety standards. In high-level biosafety laboratories, the economic and time costs of highly pathogenic virus experiments are very high. At present, there are not many P3 and P4 laboratories in my country, and these laboratories have high operating requirements and long personnel training time, resulting in slow progress in research work. Biosafety issues have always been a difficult problem that has received much attention, and the demand for high-level biosafety laboratories has also become a bottleneck for basic and applied research on FMDV. Therefore, it is particularly urgent and important to establish a pseudovirus system that can be operated in a low-level biosafety laboratory, simulate the infection process of FMDV, and is not pathogenic.

[0004] TrVLP refers to virus-like particles with transcription and replication capabilities [Furuyama, Wakako et al. "Development of an imaging system for visualization of Ebola virus glycoprotein throughout the viral lifecycle." Frontiers in microbiology vol. 131026644. 3 Nov. 2022, doi: 10.3389 / fmicb. 2022.1026644], which are produced by a biosafety level 2 (BSL-2) cell culture system, which consists of two parts [Wang, Zhongyi et al. "A RapidScreen for Host-Encoded miRNAs with Inhibitory Effects against Ebola Virus Using a Transcription-and Replication-Competent Virus-Like Particle System." International journal of molecular sciences vol. 19, 5 1488.16May.2018,doi:10.3390 / ijms19051488]: A production cell line containing genomic viral RNA (replicons) with missing nucleocapsid genes or other key genes and expressing the missing gene proteins. TrVLP can realize the complete viral life cycle of the original virus particle and is completely confined to the cells where it is produced. Specifically, trVLP can be propagated and passaged in packaging cells, while only a single round of infection can be achieved in wild-type cells, thus realizing the desire to perform biosafety downgrade treatment for some highly pathogenic viruses. The TrVLP cell culture system can be used to study the interaction between viruses and host cells, the cell tropism of viruses, the detection of neutralizing antibodies and vaccine evaluation, the screening of specific receptors, the study of the mechanism of virus entry into cells, the screening of antiviral drugs, etc. It is a very useful virological tool for emerging and re-emerging viruses. They are characterized by safety and versatility. Summary of the invention

[0005] The purpose of the present invention is to provide a foot-and-mouth disease pseudovirus cell culture system and its application. The system is a biosafety level 2 (BSL-2) cell culture system and can be used to produce foot-and-mouth disease pseudovirus with transcription and replication capabilities.

[0006] In order to achieve the above object, the present invention adopts the following technical means:

[0007] The invention discloses a foot-and-mouth disease pseudovirus cell culture system, which is composed of a subgenomic replicon FMDV-Nluc in which the Nluc gene replaces the foot-and-mouth disease virus capsid protein P1 gene and a BHK-21 cell line in which the FMDVP12A3C gene is stably integrated.

[0008] Among them, preferably, the foot-and-mouth disease virus is type O FMDV.

[0009] Preferably, the nucleotide sequence of the Nluc gene is as shown in SEQ ID NO.2.

[0010] Among them, preferably, the nucleotide sequence of the subgenomic replicon FMDV-Nluc is shown in SEQ ID NO.3.

[0011] Furthermore, the present invention also proposes the use of the foot-and-mouth disease pseudovirus cell culture system in producing foot-and-mouth disease pseudovirus having transcription and replication capabilities but not having the infectivity of live viruses.

[0012] Furthermore, the present invention also proposes a method for producing foot-and-mouth disease pseudovirus using the foot-and-mouth disease pseudovirus cell culture system, the method comprising the following steps: transfecting the subgenomic replicon in which the Nluc gene replaces the FMDV capsid protein P1 gene into a BHK-21 cell line stably integrated with the FMDV P12A3C gene, and when the cells show typical lesions of FMDV, freezing and thawing the lesioned cells three times, and harvesting FMDV virus-like particles, which are the foot-and-mouth disease pseudovirus.

[0013] Furthermore, the present invention also provides a foot-and-mouth disease pseudovirus, which has the ability to transcribe and replicate but does not have the infectivity of a live virus. The foot-and-mouth disease pseudovirus is produced using the foot-and-mouth disease pseudovirus cell culture system.

[0014] Furthermore, the present invention also proposes the use of the foot-and-mouth disease pseudovirus in the preparation and detection of FMDV antibodies.

[0015] Among them, preferably, the antibody is an anti-FMDV neutralizing antibody.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention proposes a foot-and-mouth disease pseudovirus cell culture system, and also proposes a method for producing FMDV-TrVLP in a BHK-21 cell line stably integrated with the FMDVP12A3C gene. The present invention replaces the FMDV structural protein P1 gene with the Nluc reporter gene to construct an FMDV subgenomic replicon FMDV-Nluc containing the Nluc reporter gene. FMDV-trVLPs can be propagated in the BHK-21 cell line stably integrated with the FMDV P12A3C gene, but can only cause a single infection in other cells, achieving biosafety degradation. The trVLP system provides a unique basic research application system for better understanding the life cycle of FMDV. Therefore, we have also established a serum neutralization experiment based on FMDV-trVLPs, which can provide great convenience for the detection of neutralizing antibodies.

[0018] The construction of the FMDV-Nluc replicon system has laid a foundation for in-depth research on the replication and translation mechanisms of FMDV, and also provided the necessary conditions for the construction of foot-and-mouth disease virus pseudoviruses. Specifically, the constructed FMDV-Nluc replicon can be used for research in the following areas: (1) Research on the replication and translation regulation mechanisms of FMDV, (2) Further improvement of the FMDV replicon expressing Nluc can be used as a replicon vector for the development of replicon vaccines; (3) Used as a foot-and-mouth disease replicon plasmid for the construction of foot-and-mouth disease pseudoviruses, etc.

[0019] In conclusion, the development of the FMDV-trVLPs system lacking the P1 gene has achieved biosafety downgrade and has great potential in both basic and applied research on foot-and-mouth disease virology. With its high biosafety, effectiveness, and ease of operation, FMDV-trVLPs can be used as a substitute for natural viruses in FMDV microneutralization tests, and can be used for clinical serum evaluation or vaccine immune effect evaluation experiments. Subsequently, a high-throughput neutralization test method will be established by optimizing and adjusting the experimental conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The figure is a schematic diagram of the point mutation process of the Stu I restriction site of the FMDV plasmid and the identification results of the related plasmids;

[0021] Among them, (A) schematic diagram of the process of mutating the Stu I restriction site of the FMDV plasmid; (B) electrophoresis of double restriction enzyme digestion of the FMDV plasmid and the ZJT plasmid NotI enzyme and EcoRV enzyme, M: DNA molecular mass standard; (C) verification electrophoresis of the intermediate plasmid of the product obtained after seamless cloning; (D) PCR verification electrophoresis of the MU intermediate plasmid after point mutation, 1-3 represent different plasmids; (E) bacterial solution PCR verification diagram of the MUFMDV plasmid, 1-10 represent different strains;

[0022] Figure 2 The schematic diagram of the design and assembly of the FMDV-Nluc replicon and the identification results of related plasmids;

[0023] Among them, (A) Schematic diagram of the genome structure of the FMDV-Nluc replicon, in which the P1 gene of FMDV is replaced by the Nluc reporter gene; (B) Schematic diagram of the process of the replicon FMDV-Nluc; (C) Electrophoresis of the Stu I enzyme and BsiW I enzyme double digestion of the MUFMDV plasmid, M: DNA molecular mass standard; (D) PCR electrophoresis of the Nluc gene fragment; (E) PCR electrophoresis of the P2P3 fragment; (F) PCR verification diagram of the bacterial solution transformed with the seamless cloning product, 1-7 represent different strains; (G) The FMDV-Nluc replicon and the Nluc expression plasmid were transfected into BHK-21 cells, and their luminescence values ​​were detected after 24 hours; the error bars represent the standard deviation of three independent experiments (n=6), ***p<0.001;, ****p<0.0001; the significance was evaluated by one-way analysis of variance;

[0024] Figure 3 The experimental method and related results of using FMDV-trVLPs to induce pathological changes in BHK-P1 cells;

[0025] Among them, (A) experimental scheme; (B) using a microscope to observe the pathological changes of cells; (C) extracting the RNA of FMDV-trVLPs and reversely transcribing it into cDNA, and detecting its gene status by PCR; (D) according to The luminescence value of FMDV-trVLPs was detected by luciferase assay; the error bars represent the standard deviation of one of three independent experiments performed in triplicate; ns. no difference; ****p<0.0001; significance was assessed by one-way ANOVA; (E) Plaque assay to detect the infectivity of FMDV-trVLPs;

[0026] Figure 4 The results of the study on genetic stability of FMDV-trVLPs;

[0027] Among them, (A) RNA was extracted from FMDV-trVLPs infected cells at P0 to P4 and reverse transcribed into cDNA; (B) Nluc reporter gene was detected during virus passage; (C) Two pairs of nested PCR primers were designed to perform two PCR amplifications on FMDV-trVLPs at P0 to P4, and the PCR products were separated by agarose gel electrophoresis; (D) The luminescence value of FMDV-trVLPs from P0 to P4 was detected by the luciferase kit; the error bars represent the standard deviation in three independent experiments; ns. No difference; , *p<0.05; , ****p<0.001. One-way ANOVA was used to evaluate the significance;

[0028] Figure 5 To establish the serum neutralization test method for FMDV-trVLPs;

[0029] Among them, (A, B) detection of the neutralizing ability of FMDV-trVLPs and FMDV serum; (C) analysis of cross-reaction between FMDV-trVLPs types; (D) verification of the repeatability of the FMDV-trVLPs serum neutralization test method; 5 serum samples with high to low serum titers were selected, and each serum was tested repeatedly 12 times. The titer of FMDV-rVLPs was consistent in these tests; (E) correlation between the neutralization titers determined by the FMDV-trVLPs serum neutralization test (PNT) and the traditional serum neutralization test (VNT). DETAILED DESCRIPTION

[0030] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0031] Example 1 Establishment of Foot-and-Mouth Disease Pseudovirus System

[0032] 1 Materials and methods

[0033] 1.1 Plasmids, cells, main reagents and instruments

[0034] The O-type FMDV full genome plasmid (shown in SEQ ID NO.1) and the Nluc gene sequence (shown in SEQ ID NO.2) were synthesized by GenScript Biotech Co., Ltd.; the ZJT plasmid was obtained by removing the ploy(A) and ploy(C) segments from the O-type FMDV full genome plasmid by restriction endonuclease Stu I, BsiW I, Not I and EcoR V enzymes and then ligating; the pcDNA3.1(+) vector was preserved by the foot-and-mouth disease prevention and control technical team of the Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences. BHK-21 cells and the BHK-21 cell line BHK-P1 with stable integration of the P12A3C gene (also known as I-L127P, recorded in the following literature: 1. Tan Shuzhen, Dong Hu, Sun Shiqi, et al. Construction of a foot-and-mouth disease virus-like particle inducible expression vector and screening of its BHK-21 cell pool [J]. Chinese Journal of Biotechnology, 2023, 39(12): 4849-4860. DOI: 10.13345 / j.cjb.230114; 2. Guo Huichen, Tan Shuzhen, Sun Shiqi, et al. A method for constructing a foot-and-mouth disease virus-like particle inducible expression vector [P]. Gansu Province: CN202310453994.1, 2023-09-19.) are preserved and provided by the Foot-and-Mouth Disease Prevention and Control Technology Team Laboratory of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences. Not I enzyme, EcoR V enzyme, Stu I enzyme, BsiW I enzyme and T4 DNA ligase were purchased from Takara Company; LipofectamineTM2000 and DMEM cell culture medium were purchased from Invitrogen Company; CO2 constant temperature cell culture incubator was purchased from Thermo Company, USA; PCR instrument was purchased from Biometra Company; fluorescence microscope was purchased from Leica Company, Germany.

[0035] 1.2 Primers

[0036] Primers were designed based on the complete genome sequence of FMDV type O and the Nluc gene sequence (Table 1) and synthesized by GenScript Biotech Co., Ltd. and Qingke Biotech Co., Ltd.

[0037] Table 1 Primers used for vector point mutation and construction of FMDV replicon

[0038]

[0039] 1. Construction of 3MU FMDV plasmid

[0040] In order to facilitate the construction of FMDV-Nluc replicon, we first modified the O-type FMDV whole genome plasmid (referred to as "FMDV plasmid"). The construction of FMDV-Nluc replicon adopts seamless cloning technology, in which the linearization of the vector is completed by the Stu I restriction site and the BsiW I restriction site; but since there are two Stu I restriction sites on the O-type FMDV whole genome plasmid, one of the Stu I restriction sites needs to be mutated to complete the linearization of the vector. The schematic diagram of the process of mutating the Stu I restriction site of the O-type FMDV whole genome plasmid is shown in the figure. Figure 1 As shown in A, there are ploy (A) and ploy (C) segments on the FMDV plasmid, so point mutations cannot be performed on the original plasmid; the mutant segment must first be constructed on the intermediate plasmid, and then the segment is reconnected back to the original plasmid after the mutation is completed to complete the transformation of the O-type FMDV full genome plasmid.

[0041] In order to assemble the intermediate plasmid, we first double-digested the ZJT plasmid and the FMDV plasmid with restriction endonucleases Not I and EcoRV. The digestion products were separated by agarose gel electrophoresis and the sizes were consistent with the expectations ( Figure 1 B), gel recovery for connection (this step can also be used with primers ZJT-F and ZJT-R, T7-F and T7-R PCR to derive the intermediate fragment and FMDV vector fragment respectively). The vector fragment cut by enzyme is connected with the intermediate fragment by T4 DNA ligase to obtain the intermediate plasmid ( Figure 1 C). Next, the intermediate plasmid was mutated at the Stu I restriction site using the point mutation primers MU-STU I-PCDNA3.1-F and MU-STU I-PCDNA3.1-R. After the mutation was completed, a pair of specific primers pcDNA-Stu IF and pcDNA-Stu IR were designed to verify the success of the mutation ( Figure 1 D), the results showed that the mutation was successful. Then the mutated fragment was reconnected to the FMDV plasmid to form the MU FMDV plasmid, and then the plasmid was transformed. Ten strains of bacteria were selected for bacterial liquid PCR verification. The results are as follows Figure 1 E. Eight samples that met the expected size were sent to Qingke Biotechnology Company for sequencing, and the sequencing results were all in line with expectations, indicating that the MUFMDV plasmid transformation was successful.

[0042] 1.4 Construction and identification of FMDV-Nluc replicon

[0043] The FMDV replicon was constructed by using seamless cloning technology to replace the FMDV structural protein P1 gene with the Nluc reporter gene ( Figure 2 A), construct the FMDV subgenome FMDV-Nluc replicon containing the Nluc reporter gene, the construction process diagram is as follows Figure 2 B. First, the MUFMDV plasmid was double-digested with restriction endonucleases Stu I and BsiW I, and the digestion products were separated by agarose gel electrophoresis ( Figure 2 C), and gel recovery was performed for later use. PCR amplification of Nluc reporter gene ( Figure 2 D) and P2P3 fragments ( Figure 2 E) for standby use. The construction of the FMDV replicon requires the connection of the FMDV vector with the Nluc fragment and the P2P3 fragment in sequence. Therefore, it is necessary to design an overlapping region with 17 base pairs overlapping the end of the other fragment at the end of the FMDV vector and the junction of the two fragments as a primer, so as to obtain two pairs of primers BGJY-F and BGJY-R, P2P3-F and P2P3-R. These two pairs of primers are used to amplify the two insertion fragments Nluc fragment and P2P3 fragment that can be homologous recombination reaction. The Nluc fragment and the P2P3 fragment are mixed with the double-enzyme-cut FMDV vector in proportion, and the recombination reaction is completed at 50°C for 15 minutes under the catalysis of Exnase (completed in a PCR instrument) to achieve the construction of the FMDV-Nluc replicon. The product is transformed, and a single clone is picked on the plate for positive screening. The bacterial solution PCR verification results show that the band sizes of samples 3, 5, 6 and 7 are in line with expectations ( Figure 2 E), the plasmids extracted from these strains were sent for sequencing, and the sequencing results showed that the FMDV-Nluc replicon was successfully constructed, and its nucleotide sequence is shown in SEQ ID NO.3.

[0044] 1.5 Conversion

[0045] Add 10 μL of FMDV-Nluc replicon to 100 μL of prepared competent cells and place on ice for 30 minutes. Heat shock at 42℃ for 90 seconds. Quickly ice bath for 3 minutes. Add 800 μL of preheated LB and gently shake at 37℃ for 1 hour. Centrifuge at 4500rpm for 3 minutes. Discard part of the supernatant and keep 100-300 μL to spread on LB solid culture medium containing corresponding antibiotics. Invert at 37℃ and culture for 12-16 hours. Pick a single colony into the corresponding resistance LB, shake and culture at 220rpm and 37℃ overnight for the next step of plasmid extraction and identification.

[0046] 1.6 Plasmid extraction

[0047] Pick a single colony on the plate and place it in the corresponding resistance LB for expansion culture. Take 5mL of overnight culture solution, centrifuge at 12,000rpm for 2 minutes, and discard the supernatant. Use 250μL of SolutionⅠ containing RNaseA to fully suspend the bacterial precipitate. Add 250μL of SolutionⅡ and gently flip up and down to mix 5-6 times to fully lyse the bacteria and form a transparent solution. Add 350μL of 4℃ pre-cooled SolutionⅢ, gently flip up and down to mix 5-6 times, and then let it stand at room temperature for 2 minutes. Centrifuge at 12,000rpm for 10 minutes at room temperature and take the supernatant. Transfer the supernatant to the Spin Column, centrifuge at 12,000rpm for 1 minute, and discard the filtrate. Add 500μL of BufferWA to the Spin Column, centrifuge at 12,000rpm for 30 seconds, and discard the filtrate. Add 700μL of BufferWB to the Spin Column, centrifuge at 12,000rpm for 30 seconds, discard the filtrate, and repeat this operation step. Place the Spin Column back on the Collection Tube and centrifuge at 12,000 rpm for 1 minute to remove all residual wash solution. Place the Spin Column on a new 1.5 ml centrifuge tube, add 50 μL of sterile water to the center of the Spin Column membrane, and let stand at room temperature for 1 minute. Centrifuge at 12,000 rpm for 1 minute to elute the DNA.

[0048] 1.7 Plasmid transfection

[0049] 12-well cell culture plates were plated, and transfection was started when BHK-P1 cells grew to 80%, and blank controls and empty vector transfection controls were set up. The ratio of FMDV-Nluc plasmid to liposome was μg:μL=1:2; 1.5mL centrifuge tubes were placed on the test tube rack, and 100μl Opti-MEM was added to each tube; 1.5μg plasmid was added to the first centrifuge tube, and the mixture was mixed by blowing gently and then allowed to stand; 3μL liposomes were added to the second centrifuge tube, and the mixture was mixed by blowing gently and then allowed to stand; after 4-5 minutes, the liquids in the two centrifuge tubes were mixed, and the mixture was mixed by blowing gently and then allowed to stand for 20 minutes; the original culture medium in the culture plate was discarded, and after washing twice with PBS, 600μL of cell culture solution containing 2% FBS preheated at 37°C was added to each well, and the mixture of plasmid and liposome was added drop by drop, and the culture plate was gently shaken to mix the liquid thoroughly, and then placed in a cell culture incubator for incubation.

[0050] 1.8 In mammalian cells Luciferase detection (lysis method)

[0051] Before testing, equilibrate the reagents and samples to room temperature, take out the 96-well cell plate with FMDV-trVLPs from the 37°C, 5% CO2 incubator, and leave it at room temperature for 5-10 minutes. Luciferase detection reagent and mix well. Wait for more than 3 minutes and detect the luminescent signal with an enzyme reader.

[0052] 1.9 Virus titer detection

[0053] BHK-P1 cells were plated in a 96-well plate and grown to 80% for later use. Virus solution or FMDV-trVLPs were serially diluted. The dilution was first dispensed into 10 centrifuge tubes, 0.9 ml per tube. 0.1 ml of virus suspension was added to the first tube and shaken to 10. -1 ; Take the second centrifuge tube and add 0.1ml of 10 -1 After adding the virus solution and mixing thoroughly, -2 , so diluted to 10 -10 . Pour out the culture medium in the 96-well plate, take the diluted virus solution and inoculate it into the cell monolayer, 0.1 ml per well, and repeat 8 times. 10 -1 ~10 -10 Inoculate 8 wells for each dilution, and take another virus stock solution to inoculate in a single well of cells, 0.1ml per well, as a virus control (repeat 4 wells). Take the cell culture medium and inoculate in a single well of cells, 0.1ml per well, as a normal cell control (repeat 4 wells). After inoculation, put them together in a 37°C incubator for adsorption for 1 to 1.5 hours. After adsorption, pour out the virus solution or dilution solution, and add 0.1mI cell maintenance solution to each well of cells. Culture in a 37°C cell culture incubator. Observe continuously for 24-72 hours, observe the negative control of normal cells and the positive control of the virus stock solution, and then observe and record the results of each well of the test. The results of 72 hours of observation are the final results. The results are calculated according to the Reed-Muench method. Reed-Muench formula: log-effect dose (LD 50 , ID 50 or TCID 50 = logarithm of the reciprocal of the high critical dilution of 50% infection + distance ratio × logarithm of the dilution factor. Where: distance ratio = (50% critical infection rate - 50%) / (50% critical infection rate - 50% low critical infection rate)

[0054] 1.10 Data Analysis

[0055] All statistical analyses were performed using GraphPad Prism 9, and the significance level was determined by one-way ANOVA. The results are expressed as mean ± standard deviation (SD).

[0056] 2 Results

[0057] 2.1 Identification of FMDV-Nluc replicons

[0058] In order to test the expression of FMDV-Nluc replicon, it was transfected into BHK cells and 24 h later, The luciferase detection kit detected the luminescence value of Nluc, and a positive control of Nluc and a negative control of empty load were set. The results showed that the luminescence value of the replicon was significantly different from that of the blank control, indicating that the replicon was expressed in BHK cells.

[0059] 2.2 Production and identification of FMDV-trVLPs

[0060] BHK-21 cells are susceptible to FMDV and have a high tolerance to FMDV infection. In order to explore the proliferation potential of the recombinant FMDV-Nluc replicon with P1 gene deletion in cells trans-supplied with viral P1 protein, we intend to transfect the FMDV-Nluc replicon into BHK-P1 cells to produce FMDV-trVLPs and identify them by luminescence value detection, gene detection, plaque assay and TCID50 determination ( Figure 3 A).

[0061] The FMDV-Nluc replicon was transfected into BHK-21 cells and BHK-P1 cells respectively. After 24 hours, BHK-21 cells were observed under a microscope to be in good condition, while BHK-P1 cells showed rounded and wrinkled lesions, which is a typical lesion of FMDV (CPE). Figure 3 B). The diseased cells were frozen and thawed three times and recorded as P0FMDV-trVLPs. Then, P0 FMDV-trVLPs were inoculated into BHK-P1 cells and their luminescence values ​​were detected at 24h, 36h, and 48h respectively ( Figure 3 C), the results showed that there was no significant difference in the luminescence values ​​detected at different times. P0 FMDV-trVLPs were further verified at the gene level, and their RNA was extracted and reverse transcribed into cDNA, and then PCR was performed for verification. The results are as follows Figure 3 As shown in D, the FMDV-Nluc gene was present in P0 FMDV-trVLPs, indicating that the cytopathic effect was caused by FMDV-trVLPs. Similarly, the plaque assay was used to verify ( Figure 3 E), the study found that -6 This result indicates that P0 FMDV trVLPs are pathogenic in the BHK-P1 cell line. 50 The results showed that the TCID of FMDV-trVLPs in BHK-P1 cell line 50 =10 -5.5 .

[0062] 2.3 Study on genetic stability of FMDV-trVLPs

[0063] Next, we studied the genetic stability of FMDV-trVLPs. To this end, we serially passaged FMDV-trVLPs in BHK-P1 cells, defined the FMDV-trVLPs produced by transfecting the FMDV-Nluc replicon into BHK-P1 cells as P0, and defined the cell cultures collected in subsequent passages as P1 to P4 ( Figure 4 A). RNA was extracted from each generation of FMDV-trVLPs and reverse transcribed into cDNA, which was then verified by PCR. The results were as follows Figure 4 As shown in B, as the number of passages increases, the gene level in FMDV-trVLPs decreases. Subsequently, two pairs of nested PCR primers were designed to perform two PCR amplifications on FMDV-trVLPs of passages P0 to P4. The results of the second PCR amplification are shown in Figure 4 As shown in C, the genome in the FMDV-trVLPs is continuously inherited. In order to examine the expression of the FMDV-Nluc replicon during the passage process, FMDV-trVLPs of different generations were inoculated into BHK-P1 cells, and their luminescence values ​​were detected after 24 hours. The results showed that ( Figure 4 D), only the luminescence values ​​of P0 and P1 were significantly different from those of the blank group. As the number of passages increased, the luminescence values ​​became lower and lower.

[0064] Example 2 Establishment of FMDV-trVLPs serum neutralization test method

[0065] In order to verify the application of FMDV-trVLPs in serum antibody neutralization test, we tested the neutralization ability of FMDV-trVLPs and its cross-reactivity between types. The method is as follows:

[0066] Dilute the virus solution or FMDV-trVLPs with TCID50 measured into a virus suspension with 200 TCID50. Make serial multiple dilutions of serum (pre-inactivated at 56°C for 30 min) in a 96-well microplate. First add 50 μL of growth solution to the 96-well plate, then add 50 μL of serum to be tested, mix well, and then pipette 50 μL to the next well, and so on. Until 1:256), make 4 wells for each dilution. Add 50 μL of diluted virus solution to each well, mix well, and place in a 37°C 5% CO2 incubator for 45-60 min. At the same time, set up serum toxicity control to be tested, negative and positive serum control, virus control and normal cell control, among which the virus control should be made into 4 different concentrations of 200TCID50, 20TCID50, 2TCID50, and 0.2TCID50. After completion, add 50 μL of cell suspension to each well, place in a 37°C 5% CO2 incubator, observe for 3 days and record the results. The results were calculated according to the Reed-Muench method.

[0067] The results are as follows Figure 5 As shown in A and B, O type serum

[0068] O-type FMDV-trVLPs and O-type FMDV have neutralizing ability. The infection rate of FMDV-trVLPs changes accordingly with the dilution of serum, while negative serum has no neutralizing ability. This experiment preliminarily proves the feasibility of using O-type FMDV-trVLPs instead of O-type FMDV for serum neutralization test.

[0069] There are seven serotypes of foot-and-mouth disease virus, and there is no cross-reaction between them. Only type O and type A have a wide distribution and high incidence of foot-and-mouth disease virus. In order to test whether the type O FMDV-trVLPs in this study have no cross-reaction relationship like the original virus, type A positive serum was neutralized with type O FMDV-trVLPs and type A FMDV. Figure 5 C shows that type A serum has no neutralizing ability against type O FMDV-trVLPs, and there is no cross-reaction between the two types.

[0070] In order to verify the repeatability of the FMDV-trVLPs serum neutralization test, we selected five serum samples with different titers (high, medium, and low). All serum samples were subjected to serum neutralization tests using FMDV-trVLPs. Each serum sample was tested 12 times individually, with an average intra-assay coefficient of variation (CV) of 0.65% ( Figure 5 D). The statistical results showed that the serum titer of each serum was controlled within two dilutions when the test was repeated 12 times, indicating that the pseudovirus neutralization test method has good repeatability.

[0071] Finally, to verify the correlation between the neutralizing antibody titers determined by the FMDV-trVLPs serum neutralization test (PNT) and the traditional serum neutralization test (VNT), we used a total of 20 bovine serum samples after O-type foot-and-mouth disease virus challenge and 102 bovine serum samples after O-type foot-and-mouth disease virus-like particles immunization to perform quantitative determination using both the traditional microneutralization test and the FMDV-trVLP neutralization test. The neutralizing antibody titer was determined by microscopic examination of CPE, and the highest dilution that completely inhibited CPE (ID 100 ) to indicate the neutralizing antibody titer in the serum. The results showed that the neutralization titers determined by the two methods were highly correlated, with an R square value of 0.8893 (P<0.0001) ( Figure 5 E) It is important to note that neutralization titers measured by different laboratory assays vary and need to be calibrated using the same serum panel in order to be comparable.

Claims

1. A foot-and-mouth disease pseudovirus cell culture system, characterized in that: The cell culture system consists of a subgenomic replicon FMDV-Nluc in which the Nluc gene replaces the capsid protein P1 gene of the foot-and-mouth disease virus (FMDV) and a BHK-21 cell line in which the FMDV P12A3C gene is stably integrated.

2. The foot-and-mouth disease pseudovirus cell culture system according to claim 1 or 2, characterized in that: The foot-and-mouth disease virus is type O foot-and-mouth disease virus.

3. The foot-and-mouth disease pseudovirus cell culture system according to claim 2, characterized in that: The nucleotide sequence of the Nluc gene is shown in SEQ ID NO.

2.

4. The foot-and-mouth disease pseudovirus cell culture system according to claim 1, characterized in that: The nucleotide sequence of the subgenomic replicon FMDV-Nluc is shown in SEQ ID NO.

3.

5. Use of the foot-and-mouth disease pseudovirus cell culture system according to any one of claims 1 to 4 in producing a foot-and-mouth disease pseudovirus having transcription and replication capabilities and not having the infectivity of a live virus.

6. A method for producing foot-and-mouth disease pseudovirus using the foot-and-mouth disease pseudovirus cell culture system according to any one of claims 1 to 4, characterized in that: The method comprises the following steps: transfecting the subgenomic replicon in which the Nluc gene replaces the FMDV capsid protein P1 gene into a BHK-21 cell line stably integrated with the FMDV P12A3C gene; when the cells show typical lesions of FMDV, freezing and thawing the lesioned cells three times, and harvesting FMDV virus-like particles, which are the foot-and-mouth disease pseudovirus.

7. A foot-and-mouth disease pseudovirus, characterized in that The foot-and-mouth disease pseudovirus has the ability to transcribe and replicate, but does not have the infectivity of a live virus. The foot-and-mouth disease pseudovirus is produced using the foot-and-mouth disease pseudovirus cell culture system described in any one of claims 1-4.

8. Use of the foot-and-mouth disease pseudovirus according to claim 7 in the preparation and detection of anti-FMDV antibodies.

9. The use according to claim 8, characterized in that The antibody is a neutralizing antibody against FMDV.

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