Respiratory syncytial virus ON1 genotype epidemic strain, fluorescent strain carrying enhanced green fluorescent protein and application of respiratory syncytial virus ON1 genotype epidemic strain

Abstract

The invention belongs to the technical field of viruses and genetic engineering, and particularly relates to a respiratory syncytial virus (RSV) ON1 genotype epidemic strain, a fluorescent strain carrying enhanced green fluorescent protein (EGFP) and application of the respiratory syncytial virus ON1 genotype epidemic strain. According to the invention, a recombinant RSV ON1 epidemic strain based on an RSV ON1 genotype strain genome and a recombinant RSV ON1 fluorescent strain carrying an EGFP tag are constructed, and a key biological material and a genetic manipulation platform are provided for research on the virulence determinant of the RSV A subtype epidemic strain; the two recombinant RSV strains can be used for researching in-vitro virus replication mechanism and pathogenesis, neutralizing antibody immune escape, cell real-time fluorescence monitoring and the like, and a technical platform is provided for research, development and evaluation of RSV vaccines, antibodies, drug screening and the like.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of viruses and genetic engineering, and particularly relates to a respiratory syncytial virus (RSV) ON1 genotype epidemic strain, a fluorescent strain carrying an enhanced green fluorescent protein (EGFP) of the RSV ON1 genotype epidemic strain and application of the fluorescent strain. BACKGROUND

[0002] RSV is the primary pathogen of lower respiratory tract infection in infants and young children worldwide, causing more than 33 million severe cases each year. RSV belongs to the Pneumoviridae family of the Paramyxoviridae family (Pneumoviridae) Orthopneumovirus ), including A and B subtypes, among which the RSV-A ON1 genotype epidemic strain causes antigenic drift due to a 72-nt repeat insertion mutation in the G gene, and the RSV-A ON1 genotype epidemic strain has become the dominant epidemic strain worldwide, accounting for more than 82% of RSV A subtype isolates. As a common respiratory pathogen, RSV has caused significant health threats to specific populations, and thus revealing the deep research of RSV infection, finding new drug targets, and developing safe and efficient vaccine products play a key role in the treatment and prevention of RSV.

[0003] Current RSV research faces three major technical bottlenecks: first, traditional virus detection relies on immunofluorescence technology or polymerase chain reaction (PCR), which requires fixation and staining or nucleic acid extraction, and cannot track the infection process in real time, and takes as long as 48-72 hours; second, existing fluorescently labeled strains are all based on the RSV Long original strain or the RSV A2 laboratory strain, and there are 37.5% amino acid differences between the G protein and the RSV-A ON1 genotype epidemic strain, leading to deviations in drug neutralization potency evaluation; third, the genome capacity of the Paramyxoviridae family is limited, and the insertion of EGFP can easily destroy essential genes for virus replication.

[0004] In some ways known to the inventors, attempts have been made to construct mCherry-labeled RSV, but the fluorescent protein is quickly lost during passage, with a loss rate of >40% at P5, and the problem of RSV-A ON1 genotype adaptability has not been solved. Therefore, there is an urgent need to develop an engineered strain that retains the specific mutations of RSV-A ON1, has genetic stability and real-time visualization functions, to break through the core technical barriers of vaccine evaluation, antiviral drug screening and infection mechanism research. SUMMARY

[0005] The application aims to provide a respiratory syncytial virus (RSV) ON1 genotype epidemic strain and a fluorescent strain carrying an enhanced green fluorescent protein (EGFP) and an application thereof, and constructs a recombinant RSV ON1 epidemic strain and a recombinant RSV ON1 fluorescent strain carrying an EGFP tag based on a genome of an RSV ON1 genotype strain, so as to provide a key biological material and a genetic operation platform for studying a virulence determinant of an RSV A subtype epidemic strain; the two recombinant RSV strains can be used for researches on a virus replication mechanism and a pathogenic mechanism in vitro, neutralizing antibody immune escape, and real-time fluorescent monitoring of cells, and also provide a technical platform for research and evaluation of RSV vaccines, antibodies, and drug screening.

[0006] The application provides a recombinant vector, which comprises a basic vector and an RSV-ON1 genotype epidemic strain genome cDNA inserted into the basic vector, and a nucleotide sequence of the RSV-ON1 genotype epidemic strain genome cDNA is shown as SEQ ID NO: 1.

[0007] Preferably, the recombinant vector further comprises an enhanced green fluorescent protein gene inserted between a G gene and an F gene of the RSV-ON1 genotype epidemic strain genome cDNA.

[0008] Preferably, the basic vector comprises a pBeloBac-11 plasmid vector.

[0009] Preferably, the RSV-ON1 genotype epidemic strain genome cDNA is inserted between a Bam HI and a Xho I site of the pBeloBac-11 plasmid vector.

[0010] The application provides a recombinant cell line, which comprises the recombinant vector in the above technical solution.

[0011] The application further provides an application of the recombinant vector in the above technical solution or the recombinant cell line in the above technical solution in preparing a recombinant RSV strain.

[0012] Preferably, the application comprises the following steps: rescuing the recombinant vector in the above technical solution to obtain the RSV-ON1 genotype epidemic strain and the RSV-ON1-EGFP fluorescent strain respectively.

[0013] Preferably, the rescuing method comprises the following steps: transfecting the recombinant vector into cells for culture; centrifuging a cell suspension obtained through the culture, and transfecting a supernatant into another cell for continuous culture; and detecting rescuing conditions of the two strains of viruses.

[0014] The application further provides a recombinant RSV strain, which comprises the RSV-ON1 genotype epidemic strain and / or the RSV-ON1-EGFP fluorescent strain, and is characterized by being obtained by rescuing the recombinant vector in the above technical solution.

[0015] This invention also provides the application of the recombinant vector, recombinant cell line, or recombinant RSV strain described in the above-mentioned technical solutions in one or more of the following: 1) Screening for anti-RSV drugs and / or antibodies; 2) One or more of the following: preparation, screening, and evaluation of RSV vaccines; 3) Preparation and / or screening of RSV diagnostic reagents.

[0016] Beneficial effects: This invention provides a circulating RSV ON1 genotype strain and a fluorescent strain carrying enhanced green fluorescent protein (EGFP), along with their applications. The invention constructs a recombinant RSV ON1 circulating strain based on the genome of an RSV ON1 genotype strain and a recombinant RSV ON1 fluorescent strain carrying an EGFP tag, providing key biological materials and a genetic manipulation platform for studying the virulence determinants of circulating RSV subtype A strains. The two recombinant RSV strains can be used for studies on in vitro viral replication mechanisms and pathogenic mechanisms, neutralizing antibody immune escape, and real-time cellular fluorescence monitoring. They also provide a technical platform for the development and evaluation of RSV vaccines, antibodies, and drug screening. Specifically, the circulating RSV ON1 genotype strain was isolated from a strain circulating in China in 2019. It belongs to the circulating genotypes of recent years and has good representativeness of the ON1 branch. This branch, AD.3, is a branch type with a high prevalence in China. The sixth amino acid in the 47 amino acid sequence before and after insertion mutates from histidine to leucine. The recombinant RSV circulating strain containing this mutation can be used for studies of single-point amino acid variations.

[0017] Based on this, this invention constructs a recombinant RSVON1 fluorescent strain carrying an EGFP tag based on a popular recombinant RSV ON1 strain. This recombinant RSV ON1 fluorescent strain enables real-time fluorescence detection of live cells, allowing monitoring of the dynamic process from syncytial formation to shedding. It also enables dynamic in vivo imaging monitoring of viral progression after infection of animal models with recombinant RSV virus, which is of great significance for studying the viral mechanism of the RSV ON1 genotype. Simultaneously, the recombinant RSV ON1 fluorescent strain allows for real-time fluorescence readings, with an operation time of 10 minutes per 96-well plate, significantly improving experimental efficiency compared to existing detection methods (immunodosing and neutralization assays for viral titer determination both require 72 hours of virus culture followed by fixation and staining, with an operation time exceeding 4 hours per 96-well plate). More importantly, the recombinant RSV ON1 fluorescent strain completely retains the RSV ON1 genotype-specific G gene mutation, with a fluorescence expression rate of 100% after continuous passage. Compared to existing recombinant strains carrying EGFP fluorescent tags, which typically lose fluorescence after 5 passages, this significantly improves genetic stability.

[0018] The summary concludes that the two recombinant RSV strains can be used for subsequent research on in vitro viral replication mechanisms and pathogenic mechanisms, neutralizing antibody immune escape and real-time cell fluorescence monitoring, and also provide a pathogenic technology platform for the development and evaluation of RSV vaccines, antibodies and drug screening. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0020] Figure 1 This is a schematic diagram illustrating the construction of the full-length ON1 genotype popular strain vector and the full-length ON1 genotype fluorescent strain infectious cloning vector carrying the EGFP tag in Example 1. Figure 2 The results of immunofluorescence verification of the rRSV-ON1 epidemic strain and the rRSV-ON1-EGFP fluorescent strain after virus rescue in Example 2; Figure 3 The results of indirect immunofluorescence detection of the rRSV-ON1 epidemic strain and the rRSV-ON1-EGFP fluorescent strain after virus rescue in Example 2; Figure 4 The results of EGFP fluorescence signal stability detection in Example 3; Figure 5 The results show the neutralizing ability of the recombinant fluorescent strain in Example 4 against neutralizing antibodies and the drug screening ability of the recombinant fluorescent strain in Example 5. Figure 6 The results are from in vivo experiments using the animal model in Example 6. Detailed Implementation

[0021] The present invention provides a recombinant vector comprising a base vector and a genomic cDNA of an RSV-ON1 genotype prevalent strain inserted into the base vector, wherein the nucleotide sequence of the RSV-ON1 genotype prevalent strain genomic cDNA is shown in SEQ ID NO:1.

[0022]

[0023] In one embodiment, the recombinant vector further includes inserting an enhanced green fluorescent protein (EGFP) gene between the G and F genes of the genomic cDNA of the RSV-ON1 genotype prevalent strain. In another embodiment, the enhanced EGFP gene is inserted at 5710 bp of the genomic cDNA of the RSV-ON1 genotype prevalent strain. The enhanced EGFP gene of this invention has the accession number MN443913.1 in GeneBank. In this invention, the enhanced EGFP gene (EGFP fluorescent tag) is inserted between the coding genes of the F and G proteins, which avoids the disadvantages of significantly inhibiting downstream viral gene expression due to insertion too close to the 3' end or resulting in excessively low exogenous gene expression due to insertion near the 5' end, effectively balancing the expression efficiency and viral replication capacity of both.

[0024] As one implementation method, the base vector can be a cloning vector or an expression vector, or it can be a plasmid, bacteriophage, granule, Ti plasmid, or viral vector. In an embodiment of the present invention, the base vector is the pBeloBac-11 plasmid vector.

[0025] In one embodiment, the genomic cDNA of the RSV-ON1 genotype prevalent strain is inserted between the Bam HI and Xho I sites of the pBeloBac-11 plasmid vector.

[0026] In one embodiment, the recombinant vector further includes a regulatory element; in another embodiment, the regulatory element includes a promoter to initiate transcription of the above-mentioned cDNA sequence, a terminator to terminate transcription of the above-mentioned cDNA sequence, an enhancer sequence, or other regulatory sequences that are beneficial to cleavage or sequence stability. In one embodiment, the regulatory element includes a T7 promoter, a hammerhead ribozyme, a hepatitis D virus ribozyme, and a T7 terminator. In one embodiment of the present invention, the recombinant vector includes a T7 promoter, a hammerhead ribozyme, RSV-ON1 genotype prevalent strain genomic cDNA or RSV-ON1 genotype prevalent strain genomic cDNA with an inserted enhanced green fluorescent protein gene, a hepatitis D virus ribozyme, and a T7 terminator from the 5' end to the 3' end. A hammerhead ribozyme is an RNA element with self-cleaving catalytic activity that can automatically and precisely catalyze the cleavage of excess nucleotides, thereby producing a 5' end with the correct end that is completely consistent with the natural viral genome. The hepatitis D virus ribozyme can automatically and precisely catalyze the cleavage of excess nucleotides at the 3' end of the transcript, thereby producing a precise 3' end consistent with the natural viral genome.

[0027] The present invention provides a recombinant cell line, wherein the recombinant cell line comprises the recombinant vector described in the above technical solution.

[0028] The present invention also provides the application of the recombinant vector or recombinant cell line described in the above technical solution in the preparation of recombinant RSV strains.

[0029] In one embodiment, the application of the present invention includes: rescuing the recombinant vector described in the above technical solution to obtain a circulating strain of RSV-ON1 genotype and a fluorescent strain of RSV-ON1-EGFP, respectively. In another embodiment, the rescue method includes: transfecting the recombinant vector into cells for culture; centrifuging the cell suspension obtained from the culture, taking the supernatant and transfecting it into another cell for further culture; and detecting the rescue status of the two viruses.

[0030] This invention provides a recombinant RSV strain, including a prevalent RSV-ON1 genotype strain and / or an RSV-ON1-EGFP fluorescent strain, obtained by rescuing the recombinant vector described in the above-mentioned technical solution. The prevalent RSV-ON1 genotype strain obtained using the recombinant vector of this invention retains the specific G gene mutation of the RSV ON1 genotype; the obtained RSV-ON1-EGFP fluorescent strain exhibits genetic stability, with a fluorescence expression rate of 100% after continuous passage; and it enables real-time fluorescence detection of live cells, allowing monitoring of the dynamic process from syncytial formation to shedding, and also enabling dynamic in vivo imaging monitoring of viral progression after infection of animal models with the recombinant virus. This is of great significance for the study of the viral mechanism of the ON1 genotype and greatly improves operational efficiency.

[0031] This invention also provides the application of the recombinant vector, recombinant cell line, or recombinant RSV strain described in the above-mentioned technical solutions in one or more of the following: 1) Screening for anti-RSV drugs and / or antibodies; 2) One or more of the following: preparation, screening, and evaluation of RSV vaccines; 3) Preparation and / or screening of RSV diagnostic reagents.

[0032] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0033] Unless otherwise specified, all materials, reagents, instruments, etc. used in the following examples are commercially available.

[0034] For the routine experimental methods described in the following examples, please refer to the third edition of Molecular Cloning: A Laboratory Manual, edited by Sambrook et al. (Beijing: Science Press, 2002). For the use of instruments, please refer to the instrument operation manual.

[0035] In the examples described below, the viruses included the prevalent rRSV-ON1 strain and the rRSV-ON1-EGFP fluorescent strain. The cells included BSRT7-9 and HEp-2 cell lines.

[0036] In the following examples, the plasmids and strains were: pBeloBac-11 plasmid was purchased from Genscript Biotech Ltd., and Top10 competent cells were purchased from TransGen Biotech Ltd.

[0037] Other reagents used in the following examples: RNase-free H2O was purchased from Novizan; plasmid extraction kit was purchased from QIAGEN; DMEM medium was purchased from Hyclone Biochemicals Ltd.; fetal bovine serum was purchased from Gibco; DyLight 488 was purchased from Invitrogen; Lipofectamine TM The 3000 Transfection Reagent was purchased from Invitrogen.

[0038] Example 1 Construction of full-length ON1 genotype popular strain vectors as follows Figure 1 As shown: The copy plasmid pBeloBac-11 was selected as the original vector. An expression cassette containing the T7 promoter, hammerhead ribozymes (HHR), full-length ON1 (the cDNA sequence shown in SEQ ID NO:1), hepatitis Delta Virus Ribozyme (HDV), and T7 terminator was inserted into the vector pBeloBac-11, located after its restriction enzyme site BamHI. The recombinant plasmid was synthesized by Nanjing Genscript Biotech Co., Ltd. and named pBeloBac-ON1 after sequencing verification.

[0039] Construction of infectious clones of full-length ON1 genotype carrying EGFP tag Using a successfully constructed full-length ON1 genotype vector as a template, an enhanced green fluorescent protein gene (GenBank: MN443913.1) was inserted between the G and F genes of the full-length ON1 gene, located in the intergenic region between the two genes. The recombinant plasmid was synthesized by Nanjing GenScript Biotech Co., Ltd. and named pBeloBac-ON1-EGFP after sequencing verification.

[0040] Example 2 Rescue of infectious clonal viruses from prevalent rRSV-ON1 strains and rRSV-ON1-EGFP fluorescent strains BSRT7-9 cells were pre-treated with DMEM medium containing 10% FBS at a ratio of 2 × 10⁻⁶ cells / year.6 Cells were seeded at a density of approximately 80% per well in 6-well cell culture plates and incubated at 37°C with 5% CO2 until the cell density reached approximately 80%. The medium was then replaced with DMEM containing 2% FBS. Cell transfection was performed according to the Lipofectamine™ 3000 Transfection Reagent instructions, as follows: First, prepare a plasmid premix in one EP tube: 1.2 μg of infectious cloning plasmid (i.e., pBeloBac-ON1 or pBeloBac-ON1-EGFP successfully constructed in Example 1), 5 μL of P3000, and 250 μL of OPTI MEM; prepare a Lipo3000 premix in another EP tube: 5 μL of Lipo3000 and 250 μL of OPTI MEM. Finally, mix the premixes obtained in the above two steps, let stand at room temperature for 15 min, and add the BSRT7-9 cells to be transfected.

[0041] 120 hours after cell transfection, the entire culture plate was frozen at -80°C, and after two freeze-thaw cycles, the entire cell suspension was collected and centrifuged at 10,000×g for 1 min. The supernatant was then added to HEp-2 cells, and the cells were cultured continuously, with daily observation of cell condition. Three days after virus rescue, syncytial formation, aggregation, and shedding of cells were observed, indicating cytopathic effects (CPE). Negative control cells (Hep-2 cells not inoculated with transfected cell supernatant, no treatment) showed no significant CPE. The CPE characteristics of the prevalent rRSV-ON1 strain and the laboratory-isolated live virus strain (laboratory number: 6914) were essentially consistent.

[0042] The 10th generation salvage virus was tested using RSV anti-F protein palizumab (concentration 0.75 mg / mL, purchased from Beijing Hexiucheng Biotechnology Co., Ltd., product number 01-094-0.1 mg). The results are as follows. Figure 2 As shown, the rescued virus exhibits specific green fluorescence, while the negative control group shows no fluorescence, further verifying the successful virus rescue.

[0043] Indirect immunofluorescence assay confirmed that the CPE characteristics of the rRSV-ON1 epidemic strain and the rRSV-ON1-EGFP fluorescent strain were basically the same, and the rRSV-ON1-EGFP fluorescent strain produced green fluorescence. Figure 3 The rRSV-ON1-EGFP strain expressed enhanced green fluorescent protein. No fluorescence was produced in the prevalent rRSV-ON1 strain and the negative control group, verifying that the rRSV-ON1-EGFP fluorescent strain could spontaneously fluoresce green after successful virus rescue.

[0044] Example 3 EGFP fluorescence signal stability detection The successfully identified recombinant virus rRSV-ON1-EGFP was passaged 10 times in HEp-2 cells. After each passage, the cells were harvested after 48 hours and passaged again. The harvested, centrifuged viral supernatant was inoculated into HEp-2 cells for fluorescence signal detection. The results are as follows: Figure 4 As shown.

[0045] Depend on Figure 4 It can be concluded that: from generation P1 to P10, the cells all expressed green fluorescent protein EGFP, and the fluorescence expression did not decrease significantly; second-generation sequencing of the viral nucleic acid of generation P1, P5, and P10 did not detect any site mutations, indicating that the recombinant virus can be stably expressed during in vitro culture.

[0046] Example 4 Virus neutralization assay to determine the neutralizing ability of recombinant fluorescent strains against neutralizing antibodies: The prevalent rRSV-ON1 strain and the rRSV-ON1-EGFP fluorescent strain were incubated with serially diluted nisvirimumab (purchased from Beijing Hexiucheng Biotechnology Co., Ltd., product manufactured in the USA: Prosci Incorporated, product number PSI-10-913-0.1mg) or palizumab at 37°C for 1 hour. The serial dilution procedure was as follows: the initial concentration of nisvirimumab (3.67 mg / mL) was diluted to 5 × 10⁻⁶ mg / mL. -4 mg / mL, followed by 2-fold serial dilution.

[0047] Viral titers after incubation were determined using HEp-2 cells. Specifically, viral RNA was extracted 48 hours post-infection and reverse transcribed. The neutralization titer was then determined using a fluorescence neutralization assay. The specific steps were: the virus was seeded into 96-well Hep-2 cells, cultured for 48 hours, and then fluorescence was scanned using a fluorescence microplate reader to obtain the fluorescence count in each well. The neutralization titer was calculated. The results are shown below. Figure 5 As shown in the left and middle images.

[0048] Example 5 Anti-RSV drug screening HEp-2 cells were divided into 5×10 4 / wells were seeded in 96-well black-edged permeable plates and cultured at 37°C with 5% CO2 for 24 h until 80% confluence. The culture medium was discarded. The experimental groups were added to 2% FBS maintenance medium containing gradient concentrations of ribavirin (0.1~100 μM, 3 replicates per concentration). The positive control group was given ribavirin (10 μM). The virus control group was given only 2% FBS maintenance medium. The cell control group received no virus and no drug. After pretreatment at 37°C for 1 h, the drug solution was discarded. The rRSV-ON1-EGFP fluorescent strain (MOI=0.5, 50 μL / well) was added to each well and adsorbed at 37°C for 2 h. The virus solution was discarded and replaced with maintenance medium containing the corresponding drug concentration. The cells were cultured at 37°C for another 48 h. The EGFP fluorescence intensity (excitation 485nm / emission 535nm) was read using a microplate reader, and the efficacy was calculated according to the formula. Three independent replicate experiments were performed to ensure reliability.

[0049] The results are as follows Figure 5 As shown in the middle right figure, the rRSV-ON1-EGFP fluorescent strain can be used for virus neutralization experiments and drug screening experiments. It can be used in experiments with parizumab, nisecumab, and ribavirin. The method is simple and improves experimental efficiency.

[0050] Example 6 In vivo experiments on animal models 1. Weighing the mice BALB / c mice aged 6–8 weeks were randomly divided into three groups: an rRSV-ON1 prevalent strain group, an rRSV-ON1-EGFP fluorescent strain group, and a negative control group (MOCK), with 15 mice in each group. Mice were anesthetized with isoflurane for challenge and sampling during the experiment. For the rRSV-ON1 prevalent strain group, each mouse was challenged with 2 × 10⁶ drops of isoflurane intranasally. 6 PFU / 60μL, rRSV-ON1-EGFP fluorescent strain challenged each mouse with 1×10 drops intranasally 5 PFU / 60μL, and the negative control group was given the same volume of DMEM as a control by intranasal drops, and the mice were weighed daily.

[0051] The results are as follows Figure 6 As shown, the changes in mouse body weight after infection with the prevalent rRSV-ON1 strain and the rRSV-ON1-EGFP fluorescent strain were basically consistent. The rRSV-ON1-EGFP fluorescent strain had a lower infection titer, and the mice gradually recovered after reaching their lowest value on the second day, while the prevalent rRSV-ON1 strain had a higher infection titer, and the mice gradually recovered after reaching their lowest value on the second day.

[0052] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A recombinant vector, characterized in that, The basic vector comprises a pBeloBac-11 plasmid vector.

2. The recombinant vector of claim 1, wherein, The RSV-ON1 genotype epidemic strain genomic cDNA is inserted between the Bam HI and Xho I sites of the pBeloBac-11 plasmid vector.

3. The recombinant vector of claim 1 or 2, wherein, The recombinant cell line comprises the recombinant vector of any one of claims 1-4.

4. The recombinant vector of claim 3, wherein, 6. Use of the recombinant vector of any one of claims 1-4 or the recombinant cell line of claim 5 in the preparation of a recombinant RSV strain.

5. A recombinant cell line, characterized in that, The basic vector comprises a pBeloBac-11 plasmid vector. The RSV-ON1 genotype epidemic strain genomic cDNA is inserted between the Bam HI and Xho I sites of the pBeloBac-11 plasmid vector.

7. Use according to claim 6, characterized in that, The recombinant cell line comprises the recombinant vector of any one of claims 1-4.

6. Use of the recombinant vector of any one of claims 1-4 or the recombinant cell line of claim 5 in the preparation of a recombinant RSV strain.

8. Use according to claim 7, characterized in that, The basic vector comprises a pBeloBac-11 plasmid vector.

9. A recombinant RSV strain comprising an RSV-ONl genotype epidemic strain and / or an RSV-ONl-EGFP fluorescent strain, characterized in that, The RSV-ON1 genotype epidemic strain genomic cDNA is inserted between the Bam HI and Xho I sites of the pBeloBac-11 plasmid vector. The recombinant cell line comprises the recombinant vector of any one of claims 1-4.

10. Use of the recombinant vector of any one of claims 1-4 or the recombinant cell line of claim 5 or the recombinant RSV strain of claim 9 in one or more of the following: 1) screening of anti-RSV drugs and / or antibodies; 2) one or more of preparation, screening and evaluation of RSV vaccines; 3) preparation and / or screening of RSV diagnostic reagents.

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