Recombinant feline infectious peritonitis double-layer vesicle tracer virus as well as construction method and application thereof

Abstract

The invention discloses a recombinant feline infectious peritonitis double-layer vesicle tracer virus as well as a construction method and application thereof, and belongs to the technical field of bioengineering. A CRISPR / Cas9 mediated homologous recombination technology is used, on the basis of a reverse genetic manipulation platform, an Nsp3 region of plasmid pBAC-FIBV-791146 is replaced with a reporter gene sequence, then the feline infectious peritonitis virus is rescued, and the recombinant feline infectious peritonitis bilayer vesicle tracer virus is formed. The biological characteristics of the strain and the tracing effect of the double-membrane vesicles are measured, it is proved that insertion of the ZsGreen gene does not affect virus proliferation, but the recombinant virus has the characteristic of tracing the double-membrane vesicles, and the recombinant virus can serve as a research tool for monitoring the forming mechanism of the double-membrane vesicles and a drug screening tool for resisting the feline infectious peritonitis virus.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to a recombinant feline infectious peritonitis bilayer vesicle tracer virus, its construction method, and its application. Background Technology

[0002] Coronaviruses are a class of single-stranded positive-sense RNA viruses with a wide host range. Their replication depends on the induction of specialized membrane structures—double-layered vesicles (DMVs)—within host cells. These vesicles, enclosed by a double membrane, provide a physically isolated microenvironment for viral genome replication and transcription, which is crucial for efficient viral replication and evasion of the host's innate immunity.

[0003] Currently, research on the formation mechanism of DMV mainly relies on static imaging techniques such as transmission electron microscopy (TEM), which cannot capture the dynamic process of DMV formation. Furthermore, due to the lack of tools capable of specifically labeling and isolating DMV-related host factors under real infection conditions, systematically identifying host factors involved in DMV biosynthesis remains a significant challenge. Although some studies have induced DMV-like structures in vitro by expressing viral non-structural proteins (such as nsp3 and nsp4), this model cannot fully simulate the complex host-virus interaction environment during real viral infection.

[0004] In feline infectious peritonitis virus (FIPV) research, various recombinant viruses have been developed, such as attenuated vaccine strains constructed by deleting the ORF7a or ORF7b genes (CN117417902B, CN118460612A), and reporter viruses constructed by replacing the ORF3 gene for neutralizing antibody detection and drug screening (CN114921424B). However, a recombinant FIPV tool virus that can track the DMV formation process in real time, in situ, and dynamically, and simultaneously identify its associated host proteins, is currently lacking.

[0005] Therefore, developing a novel recombinant FIPV that can integrate live-cell imaging and proteomics analysis is of great significance for a deeper understanding of the biological mechanisms of coronavirus organelle replication, screening of key host factors, and discovery of novel antiviral targets. Summary of the Invention

[0006] The purpose of this invention is to provide a recombinant feline infectious peritonitis bilayer vesicle tracer virus, its construction method, and its application, in order to solve the problems existing in the prior art. This bilayer vesicle tracer virus, by inserting a fluorescent protein ZsGreen tag into the non-structural protein 3 (nsp3) of the virus, can achieve real-time, dynamic, and visual tracking of the bilayer vesicle (DMV) formation process induced by the virus without affecting the normal replication of the virus.

[0007] To achieve the above objectives, the present invention provides the following solution:

[0008] In a first aspect, the present invention provides a method for constructing a recombinant feline infectious peritonitis bilayer vesicle tracer virus, comprising the following steps:

[0009] Using a reverse genetics platform, the Nsp3 region of plasmid pBAC-FIPV-791146 was replaced with a reporter gene sequence, which was then used to rescue feline infectious peritonitis virus (FIP) to form a recombinant FIP ​​bilayer vesicle tracer virus.

[0010] Preferably, the Nsp3 region of the plasmid pBAC-FIPV-791146 is amino acids 173-180 of the Nsp3 region; the amino acid sequence of amino acids 173-180 is shown in SEQ ID NO.1.

[0011] Preferably, the reporter gene is a fluorescent protein gene or a luminescent reporter protein gene;

[0012] The fluorescent protein gene is ZsGreen, mNeonGreen, or mVenus; the luminescent reporter protein gene is Nanoluc.

[0013] Preferably, the nucleotide sequence of the fluorescent protein gene ZsGreen is shown in SEQ ID NO.7.

[0014] Secondly, the present invention also provides a recombinant feline infectious peritonitis bilayer vesicle tracer virus constructed by the construction method described above.

[0015] Thirdly, the present invention also provides the application of the recombinant feline infectious peritonitis bilayer vesicle tracer virus described above in the preparation of bilayer vesicle tracers or bilayer vesicle tracer virus tools.

[0016] Fourthly, the present invention also provides a bilayer vesicle tracer comprising the recombinant feline infectious peritonitis bilayer vesicle tracer virus.

[0017] Fifthly, the present invention also provides a bilayer vesicle tracing virus tool, including the recombinant feline infectious peritonitis bilayer vesicle tracing virus.

[0018] In a sixth aspect, the present invention also provides the application of the recombinant feline infectious peritonitis bilayer vesicle tracer virus, the bilayer vesicle tracer, or the bilayer vesicle tracer virus tool in screening host factors associated with bilayer vesicle biogenesis.

[0019] In a seventh aspect, the present invention also provides the use of the recombinant feline infectious peritonitis bilayer vesicle tracer virus, the bilayer vesicle tracer, or the bilayer vesicle tracer virus tool in screening drugs against feline infectious peritonitis virus.

[0020] The present invention discloses the following technical effects:

[0021] (1) Real-time dynamic visualization of DMV formation was achieved. For the first time, a tool was provided that can be used to observe the entire process of coronavirus DMV from initiation to maturity in living cells in real time. Fluorescent signals appeared several hours after infection and accumulated in the perinuclear region in a punctate form, and their spatiotemporal dynamics were consistent with the known DMV formation process.

[0022] (2) The tool has high specificity and high reliability. The ZsGreen fluorescent signal is highly colocalized with DMV markers (such as double-stranded RNA-dsRNA) and endoplasmic reticulum markers. Through immunofluorescence colocalization (co-staining with dsRNA and endoplasmic reticulum markers), live cell imaging technology and immunoelectron microscopy (IEM) technology, it was confirmed that after cells were infected with recombinant virus FIPV nsp3-ZsGreen, ZsGreen was highly colocalized with the viral replication intermediate dsRNA. The ZsGreen signal (green) can trace DMV. The ZsGreen signal (green) is colocalized with endoplasmic reticulum markers (red). The ZsGreen fluorescent signal is specifically localized on the virus-induced DMV membrane structure.

[0023] (3) It has little impact on viral replication. Growth curves and plaque experiments showed that the replication ability of the recombinant virus FIPV nsp3-ZsGreen in vitro was not significantly different from that of the wild-type virus, indicating that the insertion of the ZsGreen tag did not destroy the core function of nsp3 or the replication efficiency of the virus.

[0024] (4) It has the potential for multi-functional applications.

[0025] Mechanism studies: Provides a powerful tool for studying the molecular mechanisms of DMV biogenesis, membrane origin, and interactions with other organelles.

[0026] Host factor screening: Using the virus-binding affinity purification-mass spectrometry (AP-MS) technique, host proteins recruited to DMV assembly sites under real infection conditions can be systematically identified.

[0027] Drug screening: High-throughput screening models can be established based on changes in the intensity or distribution of fluorescence signals to discover novel anti-coronavirus compounds that can inhibit DMV formation / assembly.

[0028] This invention utilizes CRISPR / Cas9-mediated homologous recombination technology. Based on a reverse genetics platform, eight amino acid sequences in the Nsp3 region of plasmid pBAC-FIPV-791146 are replaced with the ZsGreen sequence. The virus strain is then rescued to construct an Nsp3 gene fused with a ZsGreen tag virus. The virus rescued using the infectious clone of this invention can sustain autonomous replication and amplification, exhibits high viral titers, and stably expresses the antigen protein. Measurements of the strain's biological characteristics and its ability to trace double-membrane vesicles confirm that the insertion of the ZsGreen gene does not affect viral replication but endows the strain with the ability to trace double-membrane vesicles. This makes it a valuable tool for monitoring the formation mechanism of double-membrane vesicles and for screening drugs against feline infectious peritonitis virus (FIP). Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1 A schematic diagram illustrating the construction strategy for the recombinant plasmid pBAC-FIPV-nsp3-ZsGreen;

[0031] Figure 2 The images show the rescue and basic characterization of FIPV nsp3-ZsGreen virus. A shows cytopathic effects (bright field) in CRFK cells after infection with FIPV WT (control virus), scale bar = 100 μm; B shows fluorescence micrographs (ZsGreen green fluorescence) in CRFK cells after infection, scale bar = 10 μm; C shows nsp3-ZsGreen fusion protein expression detected by Western blotting; D shows plaque comparison with FIPV WT (control virus); E shows multi-step growth curve comparison with FIPV WT (control virus).

[0032] Figure 3 This image shows the specificity validation of FIPV nsp3-ZsGreen as a DMV tracing tool. A is a confocal microscopy image showing the high colocalization of ZsGreen signal (green) and DMV marker dsRNA (red); B is a live-cell imaging image showing real-time dynamic monitoring of ZsGreen signal (green) tracing DMV, scale bar = 5 μm; C is a live-cell imaging image showing the colocalization of ZsGreen signal (green) and endoplasmic reticulum marker (red), scale bar = 5 μm; D is a comparison image with the control virus FIPV WT showing DMV formation, scale bar = 500 nm.

[0033] Figure 4 These are the results of immunoelectron microscopy;

[0034] Figure 5 The flowchart and example results of host factor screening using FIPV nsp3-ZsGreen are shown below. A is the AP-MS experimental design flowchart, B is a Venn diagram showing the host proteins specifically enriched in the FIPV nsp3-ZsGreen infection group, C is the detection of NP protein expression level by Western blot using GAPDH as an internal control, and D is the immunofluorescence analysis with scale bar = 200µm. Detailed Implementation

[0035] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0036] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0037] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0038] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0039] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0040] In the following examples, the plasmid pBAC-FIPV-791146 (the 791146 gene was constructed into a BAC vector; the full-length genome sequence of the 791146 gene is accessed in GenBank under accession number MW030109.1) was obtained from Professor Peng Guiqing of Huazhong Agricultural University. 293 cells and CRFK cells were purchased from the American Type Culture Collection (ATCC). Optional reporter gene sequences in these embodiments include, but are not limited to, fluorescent protein genes (ZsGreen, mNeonGreen, or mVenus) and luminescent reporter protein genes (Nanoluc). This invention uses the fluorescent protein gene ZsGreen as an example for subsequent experiments.

[0041] Example 1: Construction of recombinant plasmid pBAC-FIPV nsp3-ZsGreen

[0042] A schematic diagram of the construction strategy for the recombinant plasmid pBAC-FIPV nsp3-ZsGreen is shown below. Figure 1 The specific operating method is as follows:

[0043] 1) Insertion Site Selection and Design: By comparing the nsp3 protein sequences of FIPV type I strain QS-79 and FIPV type II strain 79-1146, an 8-amino acid insertion was found in the nsp3 of strain 79-1146 (amino acids 173-180, amino acid sequence as shown in SEQ ID NO.1, SEQ ID NO.1: NVLTPAVN; corresponding nucleotide sequence as shown in SEQ ID NO.2). Structural prediction indicated that this region is located in the exposed flexible loop, and clinically, FIPV type I strain QS-79 often lacks this region. Therefore, this site was selected as the insertion location for the exogenous tag to minimize the impact on the normal folding and function of nsp3.

[0044] SEQ ID NO. 2: AATGTTTTAACTCCTGCTGTAAAC.

[0045] 2) Design and synthesize left and right homologous arm primers for homologous recombination, which contain sequences overlapping at both ends of the ZsGreen coding sequence. The ZsGreen gene is derived from a commercial vector (Addgene, #67975). The primer sequences are shown in SEQ ID NO. 3-4.

[0046] SEQ ID NO.3: GTTTCGCTCGTAGAAGAAAATtggcccagtccaagcacggcctgaccaa;

[0047] SEQ ID NO. 4: GAGGAAAGTTGCTCATCAGGgggcaaggcggagccggaggcgat.

[0048] 3) Using a BAC plasmid containing the full-length FIPV genome (e.g., pBAC-FIPV-79-1146) as a template, sgRNAs targeting the insertion sites were designed using the CRISPR / Cas9 system via the online website http: / / crispor.tefor.net / (the nucleotide sequences of the sgRNAs are shown in SEQ ID NO. 5-6 below). The fused sgRNAs were transcribed. The pBAC-FIPV-79-1146 plasmid was digested with CRISPR-Cas9 enzyme and the transcribed sgRNA (digestion system: Buffer 5 μL; plasmid 2 μg; sgRNA 1 μL each; enzyme 2 μL; water added to 50 μL; digestion at 37℃ for 5 h). The digestion products were recovered to obtain the linearized BAC vector.

[0049] sgRNA-F: 5'-GGCGATGTAATTGTCATTGC-3' (SEQ ID NO.5);

[0050] sgRNA-R: 5'-GGTTTTAGTTAAAACGTTTG-3' (SEQ ID NO. 6).

[0051] 4) Mix the linearized BAC vector fragment, the PCR-amplified ZsGreen fragment, and the homologous recombination enzyme, and perform an in vitro homologous recombination reaction (reaction system: Buffer 4μL; linearized BAC vector 200ng; fragments 100ng each; ligase 1μL; add water to 20μL; 37℃ 60min).

[0052] 5) Transform the recombinant product obtained in step 4) into competent cells (such as DH10B), incubate on ice for 30 min, heat shock at 42°C for 90 s, incubate on ice for 3 min, and screen for positive clones on LB plates containing chloramphenicol.

[0053] 6) Extract positive clone plasmids, verify the correct insertion of the ZsGreen gene by PCR amplification and sequencing, and obtain the recombinant plasmid pBAC-FIPV nsp3-ZsGreen.

[0054] 7) The pBAC-FIPV nsp3-ZsGreen recombinant plasmid was sequenced and identified. The recombinant plasmid was used as a template to amplify the ZsGreen fragment by PCR. The PCR product was sent to Qingke Biotechnology Co., Ltd. for sequencing. The sequence results were consistent with the original sequence, indicating that the construction was successful.

[0055] The nucleotide sequence of ZsGreen (SEQ ID NO.7):

[0056] Example 2: Rescue and Culture of Recombinant Virus FIPV nsp3-ZsGreen

[0057] 1) The correctly identified pBAC-FIPV nsp3-ZsGreen plasmid (2 μg) and the control plasmid pBAC-FIPV-79-1146 (2 μg) were mixed with the transfection reagent and transfected into HEK-293T cells in good growth condition (density of about 80%).

[0058] 2) 48 hours after transfection, the cell culture supernatant was collected and subjected to repeated freeze-thaw cycles at -80℃ / 37℃, and centrifuged to remove cell debris.

[0059] 3) The supernatant was seeded onto pre-coated CRFK cells. After 24 hours, cytopathic effects (CPE) and the appearance of green fluorescence signals were observed under a fluorescence microscope. Uninfected cells were used as a negative control (MOCK). Results are shown below. Figure 2 The results showed that after transfecting cells with the recombinant plasmid and the control plasmid, the supernatant was seeded into CRFK cells, and both cells showed obvious cytopathic effects, indicating that the recombinant virus was successfully rescued.

[0060] 4) When the cells show extensive CPE (about 80%), collect the cell suspension, repeatedly freeze and thaw, centrifuge and collect the supernatant to obtain the first generation virus stock solution, denoted as FIPV nsp3-ZsGreen. The first generation virus stock solution obtained from the control plasmid group is denoted as FIPV WT. They are aliquoted and stored at -80℃.

[0061] Example 3: Identification of the basic characteristics of the recombinant virus FIPV-nsp3-ZsGreen

[0062] 1) Western Blot validation of the expression of the nsp3-ZsGreen fusion protein

[0063] CRFK cells were infected with recombinant FIPV nsp3-ZsGreen and viral FIPV WT at MOI=1 for 12 hours, and cell lysates were collected. Western blotting was performed using anti-ZsGreen, FIPV NP, and GAPDH antibodies. Cells without viral infection served as the negative control (Mock), cells infected with viral FIPV WT served as the positive control (FIPV WT), and GAPDH was used as an internal control.

[0064] The results showed that cells infected with the recombinant virus should show a specific band (nsp3-ZsGreen fusion protein) at approximately 190 kDa, while no such band was observed in uninfected or wild-type virus-infected controls. Figure 2 (C)

[0065] 2) Growth kinetics measurement

[0066] Recombinant virus FIPV-nsp3-ZsGreen and viral FIPV WT were used to infect CRFK cells at MOI=0.1. Supernatants were collected at different time points post-infection (6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, and 72 hours) and analyzed using TCID45. 50 Alternatively, the viral titer can be determined using the plaque assay, and a growth curve can be plotted. Uninfected cells can be used as a negative control (MOCK).

[0067] The results show: See Figure 2 In the DE, there was no significant difference in growth trend and peak titer between the recombinant virus FIPV-nsp3-ZsGreen and the control virus FIPV WT (p>0.05).

[0068] 3) ZsGreen is positioned for DMV membrane structure verification

[0069] Using immunofluorescence co-localization (co-staining with dsRNA and endoplasmic reticulum markers), live-cell imaging, and immunoelectron microscopy (IEM), it was confirmed that ZsGreen fluorescence signals were specifically localized on virus-induced DMV membrane structures. Uninfected cells were used as a negative control (MOCK). The specific procedures are as follows:

[0070] Immunofluorescence co-localization method: Recombinant virus FIPV-nsp3-ZsGreen and virus FIPV WT were infected with CRFK cells at MOI=5 for 9 hpi, then the cells were fixed and stained with anti-dsRNA antibody.

[0071] Live cell imaging detection method: After infecting CRFK cells with recombinant virus FIPV-nsp3-ZsGreen at MOI=10 for 4 hours, the cell nuclei were stained with Hoechst 33342, and then high-content confocal live cell observation was performed for 10 hours.

[0072] Immunoelectron microscopy detection method: CRFK cells were infected with recombinant virus FIPV-nsp3-ZsGreen at MOI=5 for 9 hours and then observed under an immunoelectron microscope.

[0073] The results show: See Figure 3 AC and Figure 4 Confocal microscopy revealed a distinct yellow co-localization signal, indicating that ZsGreen highly co-localizes with the viral replication intermediate dsRNA. Real-time dynamic monitoring using live-cell imaging showed that the ZsGreen signal (green) could trace DMV, and that the ZsGreen signal (green) co-localized with endoplasmic reticulum markers (red). Immunoelectron microscopy revealed that the ZsGreen fluorescent signal was specifically localized on the virus-induced DMV membrane structure.

[0074] 4) DMV formation capability determination

[0075] CRFK cells were infected with recombinant virus FIPV-nsp3-ZsGreen and virus FIPV WT at MOI=5, respectively. The ability of recombinant virus and control virus FIPV WT to induce DMV formation was monitored at different time points (3hpi, 6hpi, 9hpi, 12hpi) by transmission electron microscopy.

[0076] The results showed that the ability of recombinant viral FIPV-nsp3-ZsGreen and viral FIPV WT to induce DMV formation in CRFK cells was consistent at different time points. Figure 3 (D).

[0077] Example 4: Host factor screening using FIPV nsp3-ZsGreen

[0078] See the screening flowchart Figure 5 A. The specific steps are as follows:

[0079] 1) Large-scale CRFK cell culture was used for infection experiments, divided into four groups: Mock (blank control), FIPV WT infection group (negative control), FIPV deORF3-ZsGreen infection group (negative control), and FIPV nsp3-ZsGreen infection group (experimental group). Among them, the strain of the negative control FIPV deORF3-ZsGreen infection group was FIPV deORF3-ZsGreen, which was constructed by replacing the ZsGreen tag with the ORF3 region for recombinant virus rescue, and served as the negative control strain of the ZsGreen tag.

[0080] 2) Lyse cells and perform immunoprecipitation (IP) on the lysate using anti-ZsGreen agarose beads.

[0081] 3) Elute the IP complex, perform trypsin digestion, and then perform proteomics analysis using liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0082] 4) Compare the mass spectrometry data of the four groups of samples to screen for host proteins that are specifically enriched in the FIPV nsp3-ZsGreen group, but not enriched or have very low enrichment levels in the other three groups. Figure 5 (B). These proteins are potential host factors associated with nsp3 / DMV.

[0083] Based on the above screening method, the DMV-related host factors obtained through screening were knocked down or knocked out using CRISPR / Cas9 technology to obtain CRFK cells with knocked-out DMV-related host factors. FIPV nsp3-ZsGreen virus was then used to infect the gene-edited CRFK cells at MOI=0.1 for 12 hpi. Changes in viral NP nucleoprotein expression in CRFK cells were observed using Western blotting and immunofluorescence analysis. GAPDH was used as an internal control, and viruses without CRISPR / Cas9 knockdown or knockout of candidate host factor genes served as positive controls (NC group).

[0084] The results are visible. Figure 5In CRFK cells, knockdown or deletion of FLNB, FN1, DEK, CCT7, CLTC, RACK1, RUVBL1, RPS2, CDKL5, CALM3, CORO6, SLC30A7, MYO5A, MYO1E, XRCC6, PCM1, MAP1B, MACF1, RIC8A, CHD3, and CEBPB using CRISPR / Cas9 technology resulted in either a decrease or an increase in viral NP nucleoprotein expression levels. Figure 5 Immunofluorescence analysis of knockdown or knockout cells with reduced viral NP nucleoprotein expression levels indicated that viral infection was significantly inhibited in these cells. Figure 5 (D). Therefore, the FIPV-nsp3-ZsGreen tracer virus prepared using this invention can screen for DMV-related host factors, providing a basis for subsequent research on products against feline infectious peritonitis virus.

[0085] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method of constructing a recombinant feline infectious peritonitis double-layered vesicular tracer virus, characterized by, The method comprises the following steps: The Nsp3 region of the plasmid pBAC-FIPV-791146 is replaced with a reporter gene sequence by using a reverse genetic operation platform, and then a recombinant feline infectious peritonitis double-layer vesicle tracer virus is rescued.

2. The construction method of claim 1, wherein, The Nsp3 region of the plasmid pBAC-FIPV-791146 is the 173-180th amino acid of the Nsp3 region; the amino acid sequence of the 173-180th amino acid is shown in SEQ ID NO.

1.

3. The construction method of claim 1, wherein, The reporter gene is a fluorescent protein gene or a luminescent reporter protein gene. The fluorescent protein gene is ZsGreen, mNeonGreen or mVenus; and the luminescent reporter protein gene is Nanoluc.

4. The construction method according to claim 3, characterized in that, The nucleotide sequence of the fluorescent protein gene ZsGreen is shown in SEQ ID NO.

7.

5. A recombinant feline infectious peritonitis double-layer vesicle tracer virus obtained by the construction method of any one of claims 1-4.

6. Use of the recombinant feline infectious peritonitis double-layer vesicle tracer virus of claim 5 in the preparation of a double-layer vesicle tracer or a double-layer vesicle tracer virus tool.

7. A bilayer vesicle tracer, characterized in that, The recombinant feline infectious peritonitis double-layer vesicle tracer virus of claim 5.

8. A bilayer vesicle tracer virus tool, characterized in that, The recombinant feline infectious peritonitis double-layer vesicle tracer virus of claim 5.

9. Use of the recombinant feline infectious peritonitis double-layer vesicle tracer virus of claim 5, the double-layer vesicle tracer of claim 7 or the double-layer vesicle tracer virus tool of claim 8 in screening host factors related to double-layer vesicle biogenesis.

10. Use of the recombinant feline infectious peritonitis double-layer vesicle tracer virus of claim 5, the double-layer vesicle tracer of claim 7 or the double-layer vesicle tracer virus tool of claim 8 in screening drugs against feline infectious peritonitis virus.

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