CN110157685B - Preparation method and application of replication-defective west nile virus - Google Patents

Abstract

The invention belongs to the technical field of biology, and particularly discloses a preparation method and application of replication-defective west nile virus. WNV-delta NS1 replication defective virus generated by cloning West Nile virus with deletion of non-structural protein NS1 gene through NS1 cell line trans-complementation rescue has similar growth tendency and immunogenicity as wild type WNV virus and high genetic stability, can only be replicated and amplified in a screened cell line expressing WNV NS1, and can be used for vaccine-related research of the virus in a biosafety secondary laboratory. The virus can be used as a safe and efficient candidate vaccine to prevent WNV virus infection, and has good application prospect and important strategic significance for the potential threat of China to WNV.

Description

Preparation method and application of replication-defective west nile virus

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a preparation method and application of replication-defective west nile virus.

Background

West Nile Virus (WNV) belongs to the genus of flavivirus in the family of Flaviviridae, and other viruses of the same genus include dengue virus (DENV), yellow Fever Virus (YFV), zika virus (ZIKV), and the like. The transmission cycle of WNV in nature is bird-mosquito-bird, where bird is the storage host for the virus, mosquito is the main transmission vehicle, and humans and horses are the occasional hosts for the virus. West Nile fever can be caused after people are infected with WNV, and central nervous system diseases such as lethal encephalitis and meningitis can be generated secondarily. WNV was first discovered in 1937 in the west nile area of uda and was initially spread only to a small extent and did not cause serious illness until 1999 in the continental america after a sudden epidemic in new york in the united states, which caused serious public health problems. WNV poses a threat to human health, and also causes infection and death of a large number of domestic and wild birds, such as horses and birds, in North America and the like, which seriously affects the breeding industry and ecology.

WNV viruses belong to the third-level biosafety viruses, and live virus operation needs to be performed in a third-level biosafety laboratory, so that research on the viruses is greatly limited. Vaccines are an effective means of preventing WNV infection, and no WNV vaccine is currently approved for human use on the market. The research of WNV vaccines mainly focuses on inactivated vaccines, attenuated live vaccines, recombinant subunit vaccines, and DNA vaccines. Attenuated live vaccines have strong immunogenicity, can cause humoral and cellular immune responses, induce relatively comprehensive and durable immune protection, and are regarded as important in the research and development of WNV vaccines. However, the vaccines have the hidden danger of virulence reversion, and the safety is the key point of the human vaccines, and particularly, the third-class pathogen with higher biological safety level is more important.

The WNV genome is a single positive strand RNA, approximately 11kb in length. The entire genome includes a5 'Non-Coding Region (NCR), a 3' Non-Coding Region, and an Open Reading Frame (ORF). The open reading frame encodes a multimeric precursor protein which can be cleaved into 3 structural proteins by viral and host proteases: capsid protein (Capsid protein; C), membrane protein (M) and Envelope protein (E) and 7 non-structural proteins: NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5. Structural proteins play an important role in the assembly and maturation of viral particles, and non-structural proteins are mainly involved in the replication and immune escape of the viral genome. The nonstructural protein NS1 is a highly conserved multifunctional glycoprotein, has a size of about 40kD, is involved in early viral replication, and interacts with proteins NS4A, NS4B and the like. WNV NS1 protein is important for virus immune response and replication, and early studies show that WNV NS1 can interact with RIG-I and MDA5, thereby inhibiting the generation of interferon beta; the NS1 gene can affect the virus replication after mutation, and the replication reduction can be complemented in trans by providing wild type NS 1; after the large fragment of the NS1 gene of YFV is deleted, the virus can not be replicated, and the replication defect of NS1 can be effectively complemented by supplying NS1 protein in trans, so that the replication defect virus is obtained. The replication-defective virus has similar growth and immunogenicity to wild virus, can only be replicated and amplified in a screened cell line expressing a deletion gene, has the potential of being used as a safe vaccine, and is widely applied to the research and development of vaccines of high-grade biological safe pathogens in recent years.

At present, most of the flavivirus researches are carried out by sequentially transfecting a plasmid expressing a deletion gene such as NS1/NS5 and RNA expressing the NS1/NS5 gene into cells to complement and generate a defective virus. The virus titer is usually low due to the influence of transfection reagents, transfection conditions and the like, and the virus amplification needs to be carried out again for transfection each time, which is time-consuming and labor-consuming. And screening the cell line stably expressing the deleted gene can complementarily rescue the deleted gene into the cell line only by transfecting the RNA deleted with the gene, and can continuously obtain a large number of defective viruses only by infecting the rescued defective viruses with the reamplified viruses, however, the screening of a stable and efficient cell line is long in time and difficult. Applicants successfully screened 293T in previous studies to provide WNV NS1 protein in trans_NS1Cell line (a)Zhang HL,Ye HQ,Deng CL,Liu SQ,Shi PY,Qin CF,Yuan ZM,Zhang B.Generation and characterization of West Nile pseudo-infectious reporter virus for antiviral screening.Antiviral Res.2017May; 141-47.) and although this cell line complements the replication deficiency of WNV NS1 to obtain a replication-deficient virus, WNV- Δ NS1 replication-deficient virus is present on this cell lineCan only reach the highest titer of about 10⁵FFU/mL, the virus yield is low, and the cell source is not suitable for producing Vero cells, so that the research on vaccines is restricted. Therefore, vero from Vero cells capable of stably expressing WNV NS1 protein is screened_NS1The cell line can efficiently complement the WNV NS1 replication defect in a trans-form manner, obtain the high-titer WNV-delta NS1 replication defect virus, and provide an effective tool for the research and development of the replication defect virus as a candidate vaccine.

Disclosure of Invention

The invention aims to provide a preparation method of replication-defective west nile virus, the west nile virus prepared by the method has high titer, the immunogenicity of the west nile virus is the same as that of wild-type virus WT, the west nile virus is not diseased in IFNAR-/-mice, and the west nile virus has high safety.

The invention also aims to provide an application of the west nile virus prepared by the preparation method of the replication-defective west nile virus.

In order to achieve the purpose, the invention adopts the following technical measures:

a preparation method of replication-defective West Nile virus is obtained by subculturing NS 1-deleted West Nile virus on Vero/WNV-NS1, wherein the preservation number of the Vero/WNV-NS1 is as follows: CCTCC NO: C2019113.

the application of the west nile virus prepared by the preparation method of the replication-defective west nile virus comprises the steps of utilizing the prepared virus to prepare a west nile virus vaccine; or can be prepared into a reagent for researching the West Nile virus.

Compared with the prior art, the invention has the following advantages and effects:

1. the rescued WNV-delta NS1 replication-defective virus can only replicate and amplify in a screened WNV NS1 expression cell line, and vaccine-related research of the virus can be carried out in a biosafety secondary laboratory.

2. The WNV-delta NS1 replication-defective virus provided by the invention can be cultured and amplified in a large amount in an NS1 expression cell line derived from Vero cells, and a serum-free VP-SFM culture medium is used for culturing to obtain a high-titer virus, so that convenience is provided for the production of subsequent vaccines, and the cost is saved.

3. The WNV-delta NS1 replication-defective virus provided by the invention is stable in the process of passage, is not recombined, and has good genetic stability. Meanwhile, the recombinant human immunodeficiency virus (WT) has the same immunogenicity as wild virus (WT), is not diseased in IFNAR-/-mice and has high safety.

4. The WNV-delta NS1 replication-defective virus provided by the invention is safe in a C57BL/6 mouse model, and can enable an immunized mouse to generate high-level specific IgG antibody and neutralizing antibody after boosting immunity, and generate CD8 aiming at WNV E protein epitope characteristics⁺ IFN-γ⁺T cells react and provide good immune protection to mice.

5. In a C57BL/6 mouse model, the WNV-delta NS1 replication-defective virus provided by the invention can enable an immunized mouse to generate high-level specific IgG antibodies and neutralizing antibodies after single low-dose immunization, and provides good immune protection for mice infected by WT virus with lethal dose.

Drawings

FIG. 1 is a schematic diagram of construction of pACYC-WNV- Δ NS1, a WNV clone lacking the non-structural protein NS1 gene, and pBabe-WNV-NS1, a eukaryotic expression plasmid used to screen NS1 expressing cell lines.

FIG. 2 is a schematic diagram showing the generation of WNV- Δ NS1 replication-defective virus by transcomplementing rescue using NS1 cell line;

a: WNV- Δ NS1 replication-defective virus rescue schematic;

b: replication-defective WNV- Δ NS1 viruses in Vero_NS1And 293T_NS1Comparison of growth curves on the two cell lines;

c: WNV- Δ NS1 replication-defective virus growth curves were compared on serum-free DMEM medium and serum-free VP-SFM medium.

FIG. 3 is a comparison of the growth curve, immunophage morphology, structural protein size of the rescued WNV- Δ NS1 replication-defective virus of the present invention and WNV wild-type virus WT;

a: WNV- Δ NS1 replication-defective virus was compared to the WNV wild-type virus WT growth curve;

b: WNV- Δ NS1 replication-defective virus compared to WNV wild-type virus WT immune plaque morphology;

c: detecting WNV-delta NS1 replication-defective virus and WNV wild type virus WT structural protein E and Capsid by SDS-PAGE;

d: western blotting detects WNV-delta NS1 replication-defective virus and WT structural protein E and Capsid of WNV wild-type virus.

FIG. 4 shows that the rescued WNV- Δ NS1 replication-defective virus of the invention has the same immunogenicity as WNV wild-type virus WT;

a: detecting the neutralizing effect of a WNV E protein specific monoclonal neutralizing antibody FD011-1 on WNV-delta NS1 and WT virus;

b: WNV E protein specific monoclonal neutralizing antibody FD011-2 was tested for WNV- Δ NS1 and WT virus neutralization.

FIG. 5 shows the stability and safety test of WNV- Δ NS1 replication-defective virus;

a: detecting the WNV-delta NS1 virus after passage by indirect immunofluorescence;

b: high multiplicity of infection WNV-delta NS1-Gluc reporter viruses infect Vero respectively_NS1Detecting the value of Gluc signals of cells and Vero cells;

c: survival of IFNAR-/-mice vaccinated with WNV-. DELTA.NS 1 and WT.

FIG. 6 is an assessment of the humoral immune response to WNV- Δ NS1 replication-defective virus after boosting in the C57BL/6 mouse model;

a: WNV- Δ NS1 virus in C57BL/6 mouse model in the boost of immunity, challenge virus flow chart;

b: ELISA detects IgG antibody titer generated by the mice after the primary immunization and the enhanced immunization of WNV-delta NS 1;

c: antibody neutralization experiments detect the titer of neutralizing antibodies produced by mice after primary immunization and boosting of WNV- Δ NS 1;

d: (ii) the weight change of mice boosted with WNV- Δ NS1 after challenge;

e: enhancing the survival of mice immunized with WNV- Δ NS1 after challenge;

f: boosting the toxemia profile of mice immunized with WNV- Δ NS1 after challenge;

g: ELISA detection of IgG antibody titers after challenge in mice boosted with WNV- Δ NS1.

FIG. 7 is an evaluation of the humoral immune response to a single immunization of WNV- Δ NS1 replication-defective virus at different doses in a C57BL/6 mouse model;

a: a single different dose immunization and challenge flowsheet of WNV-delta NS1 virus in a C57BL/6 mouse model;

b: ELISA detects IgG antibody titer generated by the mice immunized with different doses of WNV-delta NS 1;

c: an antibody neutralization experiment detects the titer of neutralizing antibodies generated by mice immunized with WNV-delta NS1 with different doses;

d: the body weight change of mice immunized with different doses of WNV- Δ NS1 after challenge;

e: the survival of mice immunized with different doses of WNV- Δ NS1 after challenge;

f: the toxemia of mice immunized with different doses of WNV- Δ NS1 after challenge;

g: ELISA was performed to detect IgG antibody titers after challenge in mice immunized with different doses of WNV- Δ NS1.

FIG. 8 is an evaluation of the cellular immune effect of WNV- Δ NS1 virus after boosting in the C57BL/6 mouse model;

a: mice 7d and 14d CD8 following boost immunization with WNV- Δ NS1⁺ IFN-γ⁺Percentage of T cells;

b: mice 14d CD8 following boost immunization with WNV- Δ NS1⁺ IFN-γ⁺T cell flow diagram.

Detailed Description

The experimental methods such as plasmid linearization, RNA in vitro transcription, transfection, real-time PCR and the like involved in the part adopt the conventional methods in the field if no special description exists. The following are merely illustrative of several embodiments of the present invention. It is obvious that the present invention is not limited to the following embodiments, but many variations are possible. Therefore, modifications and improvements based on the disclosure of the present invention should be made by those skilled in the art within the scope of the claims of the present invention.

Example 1:

screening of WNV NS1 expressing cell line Vero by puromycin resistance Gene Using retroviral System_NS1

1. The eukaryotic expression plasmid pBabe-WNV-NS1 for screening NS1 expression cell line has the following construction method: using the existing pACYC-WNV-WT wild-type virus clone in the laboratory as a template, using pBabe-WNV-NS1-BamHI-F: 5-cgGATCCACTGCGATAGGTCCATAGCTCTCTTCACG-3', pBabe-WNV-NS1-EcoRI-HA-R: 5-ccgGAATTCTTAAGCGTAATCTGGAACATCGTATGGGTAAGCATTCACTTGTGTGGACTGC-3' as primers, carrying out PCR amplification by PrimeSTAR HS enzyme, and recovering a PCR product, wherein the PCR reaction system is as follows: 94 ℃ 2min,94 ℃ 20s,55 ℃ 10s,68 ℃ 2min,68 ℃ 10min, and 30 cycles. The recovered fragment is subjected to double enzyme digestion by BamHI and EcoRI, and is transformed into escherichia coli competent DH5 alpha after being connected with pBabe-puro treated by the same enzyme; the plasmids are identified to be correct by DNA sequencing and are named as WNV NS1 eukaryotic expression plasmid pBabe-WNV-NS1. The cloning construction is schematically shown in FIG. 1.

2. The clone pACYC-WNV-delta NS1 of which the non-structural protein NS1 gene is deleted has a sequence shown in SEQ ID NO. 1. The construction method comprises the following steps: the existing pACYC-WNV-WT wild-type virus clone in the laboratory is used as a framework, and amino acids at positions 4-298 of an NS1 gene are deleted to obtain pACYC-WNV-delta NS1. Specifically, pACYC-WNV-WT is used as a template, F1 and R1, F2 and R2 are used as primers (the primer sequences are F1: 5-caaCCTGGCAGGGTCCGCAGTTATTGCTATTT-3', R1: 5-GTGGTGGTGCGCCCAGTGTCAGCGTGCACGT-3', F2: 5-CTGACACTGGCGCACCACAGAGAGAGCGG-3', and R2: 5-acceatGCATGCTGTGTCTTGGCTGTCCCTC-3'), primeSTARHS is used for PCR amplification, a PCR product is recovered, and the obtained 2-segment PCR recovery product is used as a template, and the primers are F1 and R2, and the PCR product is recovered. The PCR reaction systems are as follows: 2min at 94 ℃, 20s at 94 ℃, 10s at 55 ℃, 3min at 68 ℃, 10min at 68 ℃ and 30 cycles. The recovered fusion fragment is subjected to double enzyme digestion by EcoNI and SphI, is connected with pACYC-WNV-WT treated by the same enzyme and then is transformed into escherichia coli competent HB101; the plasmid was identified as correct by DNA sequencing and was designated pACYC-WNV- Δ NS1, a clone lacking the non-structural protein NS1 gene. The cloning construction is schematically shown in FIG. 1.

3. The WNV clone pACYC-WNV- Δ NS1 in vitro transcribed RNA lacking the non-structural protein NS1 gene: the pACYC-WNV-. DELTA.NS 1 plasmid was linearized with XbaI, and after completion of the linearization was confirmed by 0.8% agarose gel electrophoresis, phenol chloroform extraction was performed, and finally 11. Mu.l of RNAase-free water was added to dissolve, and the concentration of DNA was measured using Thermo Scientific NanoDrop 2000 and the quality of DNA was checked by electrophoresis. Mu.g of phenol chloroform-extracted DNA was used as a template, and the RNA of recombinant WNV-. DELTA.NS 1 was transcribed using in vitro transcription kit T7mMESSAGE mMACHINE kit (Ambion) according to the kit instructions. RNA concentration was determined using Thermo Scientific NanoDrop 2000, RNA quality was checked using 0.8% freshly prepared agarose gel electrophoresis and stored at-80 ℃ until use.

NS1 expression cell line Vero_NS1The method used for screening is the same as 293T_NS1(Zhang HL,Ye HQ,Deng CL,Liu SQ,Shi PY,Qin CF,Yuan ZM,Zhang B.Generation and characterization of West Nile pseudo-infectious reporter virus for antiviral screening.AntiviralRes.2017May; 141-47.), the cell line was selected as follows: 1) Retroviral packaging for WNV NS1 expression: seeding 2X 10 in 10cm cell culture dish ⁶293T cells are used, so that the cell transfection density reaches about 80%; 2 1.5mL centrifuge tubes, labeled A and B, were prepared, wherein 400. Mu.L of 2 XHBS was added to A and 4. Mu.g pBabe-WNV-NS1+ 3.25. Mu.g pLP/VSVG + 5. Mu.g m57+ 40. Mu.L 2.5mol/L CaCl was added to B₂Supplementing sterile water to 400 mu L, blowing and uniformly mixing by using a gun, slowly adding the mixed solution in the B into the A while mixing, quickly blowing and beating the mixed solution after adding, immediately adding into cells, and replacing 6mL of new DMEM medium containing 10 percent FBS after 6-8 h; at 48h after transfection, the supernatant from the petri dish was collected, filtered through a 0.45 μm syringe filter and dispensed, 500 μ L each, and stored in a-80 ℃ freezer. Supplemented simultaneously with 6mL of fresh 10% FBS-containing DMEM medium, all supernatants were collected 72h after transfection and filtered, aliquoted and frozen. 2) Retroviral infection of Vero cells: inoculation of 2X 10 in 6-well plates per well⁵Vero cells, the cell density of the next day reaches about 40%; the collected retroviral supernatant 48h after transfection was added to Polybrene to a final concentration of 8. Mu.g/mL, incubating at room temperature for 10-15min, adding 1mL of virus solution into each well, placing in an incubator at 37 ℃ for 2-3h, changing to 2mL of DMEM medium containing 10% FBS, discarding the medium 24h after infection, and re-infecting the cells with the collected retrovirus supernatant for 72h, in the same manner as before. 3) Screening of cell lines expressing WNV NS1 protein: after 48h of secondary infection with retrovirus, fresh 10% FBS + DMEM medium containing puromycin (puromycin) at a final concentration of 5 μ g/mL was added to each well, fresh puromycin-containing medium was replaced every three days, cells from which no resistance genes were obtained were removed until the remaining cells grew into clusters, after washing the cells once with PBS, the cell clusters were visualized on a six-well plate with a pancreatin-soaked filter paper, digested for 1min, rapidly placed in a 24-well plate of 10% FBS + DMEM medium containing puromycin at a final concentration of 5 μ g/mL, and subjected to scale-up culture, the puromycin-containing medium was replaced every three days, and the monoclonal cells were sequentially scaled up into a 6-well plate and a T25 cell culture flask. 4) Verification of expression level of WNV NS1 protein-expressing cell line: monoclonal cell strains with higher NS1 expression levels were identified by indirect immunofluorescence experiments: monoclonal cell lines were expressed at 2X 10⁵Inoculating into 35mm cell culture dish, placing 3 slides in each dish, collecting the slides in the next day, adding 1mL PBS, washing for 3 times, adding 1mL-20 deg.C pre-cooled 5% acetone (dissolved in methanol) fixative in each well, and fixing at room temperature for 15min; the fixative was discarded, washed 3 times with 1mL PBS, and the slides in each well were removed to incubate the primary antibody, which was a rabbit-derived HA-Tag (CST) monoclonal antibody diluted 1-fold; after incubating the primary antibody for 1h at room temperature, washing the primary antibody for 10 times by PBS; incubating the secondary antibody at room temperature in the dark, the antibody being a 125-fold dilution of Fluorescein Isothiocyanate (FITC) conjugated goat anti-rabbit antibody (Proteintech); after the secondary antibody is incubated for 1h, washing for 10 times by PBS, taking out the glass slide, marking, dropping a small dot of 95% glycerol at each marked position, placing the cover glass with the cell surface facing downwards on glycerol drops, observing under a fluorescence microscope, and determining the positive rate of each cell. 5) The WNV clone that complemented the deletion of the non-structural protein NS1 gene by a cell line expressing the WNV NS1 protein generated WNV- Δ NS1 replication-defective virus level validation: 2X 10 of the cell strains with higher positive rate identified in the previous step⁵One cell was inoculated into 35mm cell culture dishes, and 3 cells were placed in each dishThe slides were transfected with 1. Mu.g of in vitro transcribed WNV-. DELTA.NS 1 RNA on the next day, following the same rescue procedure as described for WNV-. DELTA.NS 1 virus in example 2. One slide was collected at 24/48/72h after transfection, and indirect immunofluorescence was performed using 1-fold dilution of murine anti-E protein 4G2 monoclonal antibody as primary antibody and 1-fold dilution of FITC goat anti-mouse antibody (Proteintech) as secondary antibody, as follows. WNV- Δ NS1 virus amplification was reflected by WNV E protein positive cell amplification, while 72h of virus supernatant was harvested and WNV- Δ NS 1-deficient virus titer was determined, as in example 2 for WNV- Δ NS1 virus titer. And selecting the cell strain with fast amplification and highest titer of the complementary produced replication-defective virus as a seed cell line, and performing large-scale amplification and cryopreservation.

After the cell lines are screened, more than one cell line with high expression quantity of NS1 can be obtained, the level of replication-defective virus generated by cloning of trans-complementation deletion non-structural protein NS1 genes of the cell lines is compared, an optimal cell line is selected as a seed cell line, meanwhile, the NS1 gene is not lost by adding puromycin in the process of passage, and a large amount of cell lines are frozen and stored after being screened and identified.

The cell line has the culture characteristic of carrying puromycin resistance gene and can normally grow in a culture medium containing puromycin with the final concentration of 5 mu g/mL. Subsequent cell line culture, using 10% FBS + DMEM medium containing puromycin at a final concentration of 5. Mu.g/mL, at 37 ℃ 5%₂Culturing in an incubator.

The applicant has sent the 5 th generation of this cell line to the collection of the chinese type culture collection on 20/5.2019 under the classification name: vero/WNV-NS1, accession number: CCTCC NO, C2019113, address: wuhan university in Wuhan, china.

The Vero/WNV-NS1 is abbreviated as Vero for VERO cells of VERO_NS1。

Example 2:

a preparation method of replication-defective West Nile virus comprises the following steps:

WNV-. DELTA.NS 1 replication-defective virus rescue: in vitroTranscribed WNV- Δ NS1 RNA was transfected into NS1 expressing cell line Vero using lipofection_NS1And 293T_NS1(control): one day before transfection, 2X 10 cells were inoculated⁵A Vero_NS1And 5X 10⁵ A 293T_NS1Cells were plated in 35mm cell culture dishes to reach about 80% on the day of transfection; when transfection was performed, the medium in the dish was discarded, and 1mL of Opti-MEM was used to wash the cells once, followed by 1mL of Opti-MEM (to allow the cells to infiltrate); adding 1mL of Opti-MEM into a 1.5mL EP tube, adding 4. Mu.L of DMRIE-C (Invitrogen) (mixing the mixture before using DMRIE-C), mixing the mixture by reversing the top and the bottom, adding 1. Mu.g of WNV-. DELTA.NS 1 RNA obtained by in vitro transcription, and mixing the mixture by reversing the top and the bottom; quickly abandoning the Opti-MEM in the culture dish, and adding the mixture into the dish (the action is light, and the mixture is not blown against the cells); CO at 37 deg.C₂After 4 hours of culture in the incubator, the culture was discarded, and 2mL of DMEM medium containing 2% FBS was added thereto. Virus supernatants were collected at 72h after transfection to obtain WNV- Δ NS1 replication-defective viruses, which were stored at-80 ℃ and the rescue process of the defective viruses is schematically shown in A in FIG. 2.

WNV- Δ NS1 replication-defective virus titer assay: each well of a 24-well cell culture plate was seeded with 1X 10⁵A Vero_NS1Cells, one slide per well, and when the cell confluence reached 90%, the medium in the well was discarded, 100. Mu.l of the defective virus collected in step 1 above diluted 10-fold with 2% FBS-containing DMEM medium was added per well, and adsorbed in an incubator at 37 ℃ for 1h, with shaking sufficiently every 15 min. After completion of the adsorption, the virus solution in each well was aspirated off, 1mL of a 2% methylcellulose-containing cover was added, and the content of CO was 5% at 37 ℃₂After 36h incubation in the incubator of (1), slides were taken for indirect Immunofluorescence (IFA) detection: discarding methylcellulose, adding 1mL PBS, washing repeatedly for 3 times, adding 1mL-20 deg.C pre-cooled 5% acetone (dissolved in methanol) stationary liquid into each well, and fixing at room temperature for 15min; discarding the fixing solution, washing 3 times with 1mL PBS, taking out the slide in each well, and incubating a primary antibody, namely a mouse source 4G2 monoclonal antibody against the E protein, with the antibody being diluted by 1; after incubating the primary antibody for 1h at room temperature, washing the primary antibody for 10 times by PBS; incubating the secondary antibody at room temperature in the dark, the antibody being a 125-fold dilution of Fluorescein Isothiocyanate (FITC) conjugated goat anti-mouse antibody (Proteintech);after the secondary antibody is incubated for 1h, washing for 10 times by PBS, taking out the glass slide, marking, dropping a small point of 95% glycerol at each marked position, placing the cover glass with a cell surface facing downwards on glycerol drops, observing under a fluorescence microscope, determining the number of anti-E protein fluorescent cell clusters, and further determining the titer of the defective virus.

The defective virus titer was expressed as FFU/mL and was calculated as follows: titer (FFU/mL) = N × 10 × (SW/SIA). ( N: the number of anti-E protein positive cell clusters under a single slide field; 10n: dilution times; 10: the ratio of 100. Mu.l of virus liquid per ml to the volume of virus liquid infecting the 24-well plate; SW: surface area of 24-well plate; and (3) SIA: area of slide )

By Vero_NS1Cell line rescued replication-defective virus titers were: 1.4X 10⁸FFU/mL; by 293T_NS1The cell line rescued replication-defective virus titers were: 2.5X 10⁴FFU/mL。

WNV-. DELTA.NS 1 replication-defective Virus in Vero_NS1And 293T_NS1Comparison of the growth curves on two cell lines and on DMEM medium with serum and VP-SFM medium without serum:

two 35mm cell culture dishes were inoculated with 2X 10 cells respectively⁵A Vero_NS1And 5X 10⁵ A 293T_NS1Cells, at 37 ℃,5% CO₂Under culture conditions, when the confluency reached 60%, 400. Mu.l of the diluted WNV-. DELTA.NS 1-deficient virus was added to each dish at an MOI of 0.1, respectively, and the concentration was 5% by CO at 37%₂After 2h of adsorption in the incubator, the virus solution was discarded, 2mL of DMEM medium containing 2% serum was added to each dish, and the content of CO was 5% at 37%₂Culturing under the culture condition, collecting 400 μ l of virus supernatant at 24/36/48/72h after infection and supplementing 400 μ l of DMEM medium containing 2% serum, and storing the collected virus at-80 deg.C. The virus titer collected at different time points was determined according to the above-described defective virus titer assay and growth curves were plotted. The results are shown in FIG. 2, B, table 1: replication-defective WNV-. DELTA.NS 1 virus in Vero _NS1293T cell line_NS1The cell line grows well, and the highest titer of the virus can be reached at 48h after infection⁸FFU/mL, and at 293T_NS1The highest titer on the cell line can only reach about 10⁵FFU/mL, indicating that the selected NS 1-expressing cell line Vero_NS1Comparison with 293T_NS1Is more suitable for the growth of WNV-delta NS1 replication-defective virus, thereby being beneficial to the production of subsequent vaccines.

TABLE 1 WNV- Δ NS1 replication-defective Virus in Vero_NS1And 293T_NS1Viral titers at different time points post-infection at the same multiplicity of infection on both cell lines

As above, two 35mm cell culture dishes were inoculated with 2X 10 cells, respectively⁵Each Vero_NS1Cells, adding WNV-delta NS1 defective virus into each dish according to the MOI of 0.1, adsorbing for 2h, discarding virus solution, adding 2mL DMEM medium containing 2% serum and serum-free VP-SFM medium into each dish, collecting virus supernatant 24/36/48/72h after infection, measuring virus titer, and drawing a growth curve. Results As shown in FIG. 2C, table 2, WNV-. DELTA.NS 1 replication-defective virus was cultured in serum-free VP-SFM medium to achieve high titers of virus similar to those achieved in serum-free DMEM.

TABLE 2 WNV- Δ NS1 replication-defective Virus in 2% serum DMEM media and serum-free VP-SFM media at different time points after infection by the same multiplicity of infection

The results show that the NS1 expression cell line Vero screened by the invention_NS1Provides convenience for the production of subsequent vaccines and saves the cost.

Example 3:

comparison of the growth curves, immunophagous morphology, structural protein sizes, and immunogenicity of the rescued WNV- Δ NS1 replication-defective viruses of the invention with WNV wild-type WT:

WNV-. DELTA.NS 1 complexGrowth curves of the defective virus and the WNV wild-type virus WT were compared: in the same manner as in example 1, two 35mm cell culture dishes were inoculated with 2X 10 cells, respectively⁵A Vero_NS1Cells were cultured by adding WNV-. DELTA.NS 1-deficient virus and WNV wild-type virus WT to each dish at a MOI of 0.1, adsorbing for 2 hours, discarding the virus solution, adding 2mL of 2% serum-containing DMEM medium to each dish, collecting the virus supernatant at 24/36/48/72 hours after infection, measuring the virus titer by the method of example 1, and plotting a growth curve (A in FIG. 3). The results showed that WNV-. DELTA.NS 1 replication-deficient viruses grew similarly to the wild-type virus WT.

WNV- Δ NS1 replication-defective virus compared to WNV wild-type virus WT immunophagous morphology: each well of a 24-well cell culture plate was seeded with 1X 10⁵A Vero_NS1When the cell confluence reached 90%, the medium in the wells was discarded, 100. Mu.l of WNV-. DELTA.NS 1 diluted 10-fold with 2% FBS-containing DMEM medium and WT virus, respectively, was added, and the mixture was adsorbed at 37 ℃ for 1 hour and sufficiently shaken every 15 min. Removing the virus solution from each well after adsorption, adding 1mL of a cover containing 2% methylcellulose, and 5% CO at 37 deg.C₂Culturing for 36h in the incubator, discarding methylcellulose, adding 1mL PBS, washing repeatedly for 3 times, adding 1mL precooled fixing solution (methanol: acetone = 1) at-20 ℃ into each hole, and fixing for 15min at room temperature; the fixative was discarded and washed 3 times with 1mL PBS; add 200. Mu.l of 1-fold diluted 4G2 monoclonal antibody per well; after incubating the primary antibody for 1h at room temperature, washing the primary antibody for 3 times by PBS; 200 μ l of a 1-fold dilution of horseradish peroxidase (HRP) goat anti-mouse antibody (Proteintech) was added per well at room temperature; after incubation for 2h with the secondary antibody, the cells were washed 3 times with PBS and developed with an enhanced HRP-DAB substrate development kit (Tiangen) in the dark. Plaque morphology was observed (B in fig. 3). As can be seen from the figure, WNV-. DELTA.NS 1 replication-defective virus has a plaque morphology similar to that of wild-type virus WT.

Concentration and purification of WNV- Δ NS1 replication-defective virus and WNV wild-type virus WT: at 175-cm²In the culture flask of (2) was inoculated with 3X 10⁶Each Vero_NS1Cells, when the degree of cell confluence reached 60%, WNV-. DELTA.NS 1 and WT (each disease) were added to each flask, respectively, at a multiplicity of infection MOI of 0.14 flasks) were subjected to virus culture, and the supernatants were collected 48h after infection. The supernatant was centrifuged at 400g for 10min at 4 ℃ and 5000rpm for 20min at 4 ℃ to remove cells and debris. PEG8000 (Sigma) with the final concentration of 8% (w/v) is added into the supernatant, the mixture is evenly mixed by turning upside down, and then the mixture is precipitated at 4 ℃ overnight, and is centrifuged at 10500g at 4 ℃ for 50min, then the supernatant is discarded, and the precipitate is dissolved by PBS. Spreading a sucrose cushion prepared with PBS at a concentration of 24% (w/v) on a ultracentrifuge tube, spreading the solubilized virus on the sucrose cushion, centrifuging the virus at 105000g for 1.5h at 4 ℃ with an Optima MAX-XP ultracentrifuge, SW41 rotor, discarding the supernatant, dissolving the pellet in 50 μ L PBS, storing at-80 ℃ for use, the titer of the concentrated and purified virus is: 1.2X 10⁹FFU/mL。

WNV- Δ NS1 replication-defective virus compared to the size of the WT structural proteins of WNV wild-type virus: protein samples of the WNV-delta NS1 and WT viruses after the superseparation and purification are respectively prepared, SDS-PAGE is carried out, and then Coomassie brilliant blue is used for staining, so that a band (C in figure 3) with the size of about 55kDa and 15kDa can be seen in the two viruses, and the molecular weights of the band are respectively consistent with those of the WNV structural protein E protein and the Capsid protein. Western blotting detection using a monoclonal antibody 4G2 specific to the virus E protein and a polyclonal antibody specific to the Capsid protein all detected bands of the E protein and the Capsid protein (D in FIG. 3). The results indicate that the WNV- Δ NS1 replication-defective virus is identical in size to the structural protein of the wild-type virus WT.

WNV- Δ NS1 replication-defective virus compared to WNV wild-type virus WT immunogenicity: neutralization assay with Plaque Reduction (PRNT)₅₀) The neutralizing effect of WNV-delta NS1 and WT of WNV E protein-specific monoclonal neutralizing antibodies FD011-1 and FD011-2 were tested, respectively. Specifically, each well of a 12-well cell culture plate was seeded with 2X 10⁵Each Vero_NS1Placing a slide in each hole of the cell, when the cell confluence reaches 90%, firstly carrying out 2-fold serial dilution on the FD011-1 monoclonal antibody and the FD011-2 monoclonal antibody, respectively uniformly mixing 100 mu l of the diluted antibody with 100FFU WNV-delta NS1 and WT with the same volume, and then incubating for 1h at 37 ℃ (the final concentration of the two monoclonal antibodies is 1.6/0.8/0.4/0.2/0.1/0.05/0 mu g/mL); discard the medium in the well, add the incubated antibody-virus mixture to the corresponding well, 37Adsorbing at the temperature of 1h, and sufficiently shaking once every 15min; after completion of the adsorption, the virus solution in each well was aspirated off, 1mL of a 2% methylcellulose-containing cover was added, and the content of CO was 5% at 37 ℃₂After the cells are cultured for 36h in the incubator, the slides are collected and subjected to indirect Immunofluorescence (IFA) to detect the number of anti-E protein fluorescent cell masses in each well, and the inhibition rate of the monoclonal antibody to WNV-delta NS1 and WT under each dilution concentration is calculated: inhibition (%) = 1-number of anti-E protein fluorescent cell masses in virus after monoclonal neutralization/number of anti-E protein fluorescent cell masses in virus without monoclonal neutralization.

The results show that the FD011-1 (A in FIG. 4) and FD011-2 (B in FIG. 4) monoclonal antibodies have similar dose-dependence on WNV- Δ NS1 and WT virus neutralization effects, indicating that WNV- Δ NS1 replication-defective virus has the same immunogenicity as wild-type virus WT.

Example 4:

WNV- Δ NS1 replication-defective virus stability and safety testing:

WNV- Δ NS1 replication-defective viruses are genetically stable: the WNV-. DELTA.NS 1 virus rescued in example 2 was designated as the P0 generation in Vero_NS1The cells are continuously passaged to P15 generation, and the equivalent amount of virus supernatant of P5/P10/P15 generation is infected with Vero_NS1And general Vero cells, the IFA experiment was performed (a in fig. 5). Vero infected with different generations of supernatant as shown in the figure_NS1The cells can detect similar more positive cells, and the Vero cells can not detect the positive cells all the time, which indicates that the WNV-delta NS1 virus is stable in the passage process, is not recombined and has good genetic stability.

WNV- Δ NS1 replication-defective virus safety: the wells of the 12-well cell culture plate were inoculated with 2X 10 cells⁵Each Vero_NS1Cells and Vero cells, when the cell confluence reaches 60%, according to the MOI of 100, respectively inoculating WNV-delta NS1-Gluc reporter virus (pACYC-WNV-Gluc reporter virus clone (reporter gene Gluc is inserted between 5' UTR and Capsid) existing in the laboratory as a framework, deleting 4-298 amino acids of NS1 gene to obtain pACYC-WNV-delta NS1-Gluc, the construction process is the same as pACYC-WNV-delta NS1 in the example 1), and dividing into two groupsIs different from 0/1/2/4/6/8/12/24/36h after infection according to Pierce^TMGaussia Luciferase Flash Assay Kit (Thermo Scientific) instructions lyse cells in wells, measure Gluc signal values using a multifunctional microplate reader Varioskan Flash (Thermo Fisher), and plot (B in FIG. 5).

Vero in two cells 1 to 6h after infection as shown_NS1The cells were similar and increased somewhat, indicating that this phase was the translation phase of WNV-. DELTA.NS 1-Gluc virus. Vero from 8 to 36h after infection_NS1The value of Gluc signal in cells is obviously increased, indicating that WNV-delta NS1-Gluc virus is in Vero_NS1Normal replication in cells, but gradual reduction of the value of Gluc signal in Vero cells indicate that WNV-delta NS1-Gluc virus can not replicate in Vero cells, further indicating that WNV-delta NS1 has replication defect and can only replicate in Vero of NS1 expression cell line_NS1It can be replicated in cell lines and has safety at cellular level.

To further verify the safety of WNV-. DELTA.NS 1 replication-deficient viruses at the animal level, 10 was added separately²WNV-WT and 2X 10 of FFU⁸FFU WNV- Δ NS1 virus peritoneal inoculation six weeks old female IFNAR-/-mice (type I interferon deficient and susceptible to WNV virus infection), each group of five mice. Mice were observed daily for 28 days of survival and survival curves were plotted (fig. 5, C). The results show that the mice inoculated with the WNV-delta NS1 virus survive and have no sign of morbidity, while the mice inoculated with the WNV-WT die at the 5 th day and show the morbidity phenomena of hair irregularity, weight loss and the like from the 2 nd day. Proves that WNV-delta NS1 virus is not pathogenic in IFNAR-/-mice, has high safety and can be used as a potential candidate vaccine.

Example 5:

the WNV-delta NS1 virus prepared by the preparation method disclosed by the invention is applied to the preparation of vaccines:

this example investigated evaluation of the humoral immune effect of WNV-. DELTA.NS 1 virus after boosting in the C57BL/6 mouse model:

1. three groups of four week old female C57BL/6 mice (five per group) were individually immunized intraperitoneally at 4.8X 10⁷WNV- Δ NS1 virus and isotomes of FFUProduct PBS, and Mock set without any treatment. Performing booster immunization 28 days after immunization according to the same dosage, taking blood 14 days and 28 days after the first immunization and the booster immunization respectively, standing at 4 ℃ for 3h, centrifuging, collecting serum, inactivating at 56 ℃ for 30min, and freezing at-20 ℃ for later use.

Detection of IgG antibody titers and PRNT by ELISA, respectively₅₀Detection of neutralizing antibody titers: 1) Coating a 96-well plate with WNV-delta NS1 virus, blocking with 5% skim milk prepared with PBS, performing 4-fold serial dilution of serum from 1 to 50, incubating with the coated 96-well plate at 37 ℃ for 2H, washing the plate with PBST for 3 times, incubating with HRP goat anti-mouse antibody at 37 ℃ for 1H, developing with a two-component developing kit (Proteitech), adding 1M H, and performing reverse transcriptase (RT-PCR) assay₂SO₄After the termination, the absorbance at 450nm was measured by a multifunctional microplate reader, and the IgG antibody titer was the highest dilution when the absorbance of the serum of the immunized mouse was 2 times that of the serum of the non-immunized mouse (B in FIG. 6). 2) Each well of a 12-well cell culture plate was inoculated with 2X 10 cells⁵When the confluence degree of the cells reaches 90%, serially diluting the serum by 2 times, respectively mixing 100 mu l of diluted antibody with 100PFU WNV-WT with the same volume, and incubating at 37 ℃ for 1h; discarding the culture medium in the wells, adding the incubated serum-virus mixture into the corresponding wells, adsorbing at 37 ℃ for 1h, and shaking sufficiently once every 15min; after completion of the adsorption, the virus solution in each well was aspirated off, 1mL of a 2% methylcellulose-containing cover was added, and the content of CO was 5% at 37 ℃₂Culturing for 72h in the culture box, staining with staining solution containing 1% crystal violet and 3.7% formaldehyde after plaque formation, treating at room temperature for 30min, collecting staining solution in the well, washing the well bottom with running water, oven drying, and counting virus titer (PRNT) and neutralizing antibody titer (PRNT) of serum after neutralization at each dilution₅₀) The highest dilution of serum when the virus titer was neutralized 50% (C in fig. 6). The results show that C57BL/6 mice can generate stronger antibody response 14d after primary immunization of WNV-delta NS1, and IgG antibody titer is increased continuously, and the titer reaches the highest value after boosting, about 1 10000. The detection result of the neutralizing antibody titer is consistent with the increase trend of the IgG antibody titer, and the neutralizing antibody titer after the boosting immunization is 2 after the primary immunizationThe titer of 8d was increased about 16-fold, the titer was about 1, 1280-1. It was demonstrated that WNV-. DELTA.NS 1 immunized C57BL/6 mice produced a strong antibody response against WNV.

2. 30 days after the boosting, the mice of the other two groups except the Mock group are not treated, and the abdominal cavities of the mice of the other two groups are respectively attacked by 3 multiplied by 10⁷PFU WNV-WT. The survival condition of the mice is recorded and the weight is weighed every day, the measurement is carried out for 28 days, meanwhile, blood is taken from the orbit at the 2 nd day after the toxin attack, and the toxemia is detected by a Real-time PCR method: 5 drops of blood were taken from each mouse in 1.5ml EP tubes, 1ml Trizol was added to each well for lysis, and RNA extraction was performed according to the instructions. Real-Time RT-PCR detection was performed with One Step SYBR Prime Script PLUS RT-PCR Kit (Perfect Real Time) (TAKARA), with WNV-F: 5-. The reaction systems are as follows: reverse transcription at 42 ℃ for 5min, pre-denaturation at 95 ℃ for 10s, PCR for 40 cycles including denaturation at 95 ℃ for 5s, annealing and extension at 60 ℃ for 30s. The results showed that the control mice immunized with PBS showed a gradual weight loss at 6D after challenge (D in fig. 6), and higher levels of toxic blood were detected at 2D after challenge (F in fig. 6), showing symptoms of morbidity such as hair loss, hind limb paralysis, etc., and total death at 15D (E in fig. 6). Mice immunized with WNV- Δ NS1 and Mock mice without any treatment survived without any disease symptoms and no toxemia detected. Simultaneously, 14d and 28d IgG antibody titers of mice immunized with WNV- Δ NS1 were determined (as above), and the increase in antibody titer (G in FIG. 6) indicated that WNV successfully infected the immunized mice. The rescued WNV-delta NS1 replication-defective virus is proved to be safe in a C57BL/6 mouse model, and can be used as a vaccine to provide good immune protection for a C57BL/6 mouse.

Example 6:

the application of the WNV-delta NS1 virus provided by the invention in the preparation of vaccines is as follows:

this example investigated the evaluation of humoral immunity and protective effect of the WNV- Δ NS1 virus in a C57BL/6 mouse model after single immunization with different doses:

1. five groups of four week old female C57BL/6 mice (five per group)Only), peritoneal immunization 3X 10 respectively⁶、1.2/4.8×10⁷WNV-. DELTA.NS 1 virus of FFU and equal volume of PBS, and Mock group without any treatment. Blood was collected at 14 and 28 days after immunization, and IgG antibody titer (B in FIG. 7) and PRNT were measured by ELISA₅₀The neutralizing antibody titer (C in fig. 7) was detected (the same method as in example 5). The results show that 14d of WNV-delta NS1 immunized C57BL/6 mice with different doses can generate antibody reaction, the antibody titer is improved at 28d, and the detection result of the neutralizing antibody titer is consistent with the increase trend of the antibody titer. It was demonstrated that a single immunization against WNV-. DELTA.NS 1 resulted in a stronger antibody response against WNV in C57BL/6 mice.

2. 30 days after immunization, the mice in the four groups except the Mock group were not treated, and the abdominal cavities of the mice in the other four groups were respectively attacked by the toxin of 3 multiplied by 10⁷PFU WNV-WT. The survival of the mice was recorded and the body weight was weighed daily for 15 days, and blood was collected from the orbit at 2d after challenge and then assayed for toxemia by the fluorescent quantitative PCR method (same as example 5). The results showed that the body weight of the control mice immunized with PBS decreased sharply and died all at 7D after challenge (D and E in fig. 7), and higher toxic blood levels could be detected at 2D after challenge (F in fig. 7), showing disease symptoms such as hair loss, hind limb paralysis, etc. Mice immunized with different doses of WNV- Δ NS1 survived the Mock group without any treatment, and no signs of disease were observed and no toxemia was detected. Mice immunized with different doses of WNV- Δ NS1 were also tested for IgG antibody titers at 14d and 28d post challenge (FIG. 7, G) (as in example 5), indicating that the WNV virus successfully infected the immunized mice. Proved that the rescued WNV-delta NS1 replication-defective virus can provide good immune protection for C57BL/6 mice by single low-dose immunization, and has the potential of being used as a candidate vaccine.

Example 6:

the invention provides an application of WNV-delta NS1 virus in preparing vaccine:

this example examined the evaluation of cellular immune effects of WNV-. DELTA.NS 1 virus after boosting in the C57BL/6 mouse model:

four groups of four-week-old female C57BL/6 mice (five in each group) were immunized intraperitoneally at 19.2X 10⁷WNV-. DELTA.NS 1 viruses from FFU, two groups were not treated at all as Mock group. The mice were boosted at the same dose 28 days after immunization, and spleens of the mice in the immunized group and Mock group were collected 7 days and 14 days after the boosting, respectively, and CD8 was detected by flow cytometry⁺ IFN-γ⁺T cell proliferation: picking spleen, and washing twice in serum-free RPMI-1640 culture medium; grinding with 200 mesh metal grinding net, centrifuging, collecting splenocyte precipitate, and lysing with 1 × erythrocyte lysate at room temperature for 5min; after centrifugation, 1640 culture medium containing beta-mercaptoethanol is added to suspend cells, and the cell concentration is adjusted to be 2 multiplied by 10⁷cells/ml; stimulating lymphocytes with 1. Mu.g/ml WNV E protein polypeptide and 50ng/ml PMA + 1. Mu.g/ml IONO at 37 ℃ for 5h, respectively, while adding 1. Mu.l/ml Golgi plug; after the stimulation, the cells were washed with 2% FBS-containing PBS staining buffer, and anti-CD8 was added thereto⁺Antibody (Ms CD8a APC-Cy7, BD Pharmingen) stained at 4 ℃ for 30min; washing cells with staining buffer, adding cell fixing membrane penetrating liquid, and treating at 4 deg.C for 20min; after washing the cells with a transmembrane rinsing buffer, adding an anti-IFN-gamma antibody (Ms IFN-Gma PE-Cy7, BD Pharmingen) to stain for 30min at 4 ℃; and washing the cells by using a transmembrane rinsing buffer solution, and then suspending the cells in a staining buffer solution for flow cytometry detection. As shown in FIG. 8, A, mouse CD8 at 7d to 14d after boosting⁺ IFN-γ⁺The activation of T cells shows a growing trend. 14d after boosting, CD8 stimulated by WNV E protein polypeptide and PMA + IONO⁺ IFN-γ⁺T cells increased from 0.2% to 1% and 3.2% before stimulation, respectively, whereas the nonimmune Mock group was CD8 stimulated by WNV E protein polypeptide⁺ IFN-γ⁺T cells were consistent with pre-stimulation levels, increasing slightly from 0.2% to 1% pre-stimulation (fig. 8B) after PMA + IONO stimulation. The result shows that the rescued WNV-delta NS1 replication defective virus can generate stronger CD8 aiming at WNV E protein epitope characteristics after strengthening the immunity of C57BL/6 mice⁺ IFN-γ⁺T cell responses.

Sequence listing

<110> Wuhan Virus institute of Chinese academy of sciences

<120> preparation method and application of replication-defective west nile virus

<160> 7

<170> SIPOSequenceListing 1.0

<210> 1

<211> 10149

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

agtagttcgc ctgtgtgagc tgacaaactt agtagtgttt gtgaggatta acaacaatta 60

acacagtgcg agctgtttct tagcacgaag atctcgatgt ctaagaaacc aggagggccc 120

ggcaagagcc gggctgtcaa tatgctaaaa cgcggaatgc cccgcgtgtt gtccttgatt 180

ggactgaaga gggctatgtt gagcctgatc gacggcaagg ggccaatacg atttgtgttg 240

gctctcttgg cgttcttcag gttcacagca attgctccga cccgagcagt gctggatcga 300

tggagaggtg tgaacaaaca aacagcgatg aaacaccttc tgagttttaa gaaggaacta 360

gggaccttga ccagtgctat caatcggcgg agctcaaaac aaaagaaaag aggaggaaag 420

accggaattg cagtcatgat tggcctgatc gccagcgtag gagcagttac cctctctaac 480

ttccaaggga aggtgatgat gacggtaaat gctactgacg tcacagatgt catcacgatt 540

ccaacagctg ctggaaagaa cctatgcatt gtcagagcaa tggatgtggg atacatgtgc 600

gatgatacta tcacttatga atgcccagtg ctgtcggctg gtaatgatcc agaagacatc 660

gactgttggt gcacaaagtc agcagtctac gtcaggtatg gaagatgcac caagacacgc 720

cactcaagac gcagtcggag gtcactgaca gtgcagacac acggagaaag cactctagcg 780

aacaagaagg gggcttggat ggacagcacc aaggccacaa ggtatttggt aaaaacagaa 840

tcatggatct tgaggaaccc tggatatgcc ctggtggcag ccgtcattgg ttggatgctt 900

gggagcaaca ccatgcagag agttgtgttt gtcgtgctat tgcttttggt ggccccagct 960

tacagcttca actgccttgg aatgagcaac agagacttct tggaaggagt gtctggagca 1020

acatgggtgg atttggttct cgaaggcgac agctgcgtga ctatcatgtc taaggacaag 1080

cctaccatcg atgtgaagat gatgaatatg gaggcggcca acctggcaga ggtccgcagt 1140

tattgctatt tggctaccgt cagcgatctc tccaccaaag ctgcgtgccc gaccatggga 1200

gaagctcaca atgacaaacg tgctgaccca gcttttgtgt gcagacaagg agtggtggac 1260

aggggctggg gcaacggctg cggactattt ggcaaaggaa gcattgacac atgcgccaaa 1320

tttgcctgct ctaccaaggc aataggaaga accatcttga aagagaatat caagtacgaa 1380

gtggccattt ttgtccatgg accaactact gtggagtcgc acggaaacta ctccacacag 1440

gttggagcca ctcaggcagg gagattcagc atcactcctg cggcgccttc atacacacta 1500

aagcttggag aatatggaga ggtgacagtg gactgtgaac cacggtcagg gattgacacc 1560

aatgcatact acgtgatgac tgttggaaca aagacgttct tggtccatcg tgagtggttc 1620

atggacctca acctcccttg gagcagtgct ggaagtactg tgtggaggaa cagagagacg 1680

ttaatggagt ttgaggaacc acacgccacg aagcagtctg tgatagcatt gggctcacaa 1740

gagggagctc tgcatcaagc tttggctgga gccattcctg tggaattttc aagcaacact 1800

gtcaagttga cgtcgggtca tttgaagtgt agagtgaaga tggaaaaatt gcagttgaag 1860

ggaacaacct atggcgtctg ttcaaaggct ttcaagtttc ttgggactcc cgcagacaca 1920

ggtcacggca ctgtggtgtt ggaattgcag tacactggca cggatggacc ttgtaaagtt 1980

cctatctcgt cagtggcttc attgaacgac ctaacgccag tgggcagatt ggtcactgtc 2040

aacccttttg tttcagtggc cacggccaac gctaaggtcc tgattgaatt ggaaccaccc 2100

tttggagact catacatagt ggtgggcaga ggagaacaac agatcaatca ccattggcac 2160

aagtctggaa gcagcattgg caaagccttt acaaccaccc tcaaaggagc gcagagacta 2220

gccgctctag gagacacagc ttgggacttt ggatcagttg gaggggtgtt cacctcagtt 2280

gggaaggctg tccatcaagt gttcggagga gcattccgct tactgttcgg aggcatgtcc 2340

tggataacgc aaggattgct gggggctctc ctgttgtgga tgggcatcaa tgctcgtgat 2400

aggtccatag ctctcacgtt tctcgcagtt ggaggagttc tgctcttcct ctccgtgaac 2460

gtgcacgctg acactgggcg caccaccaca gagagcggaa agttgataac agattggtgc 2520

tgcaggagct gcaccttacc accactgcgc taccaaactg acagcggctg ttggtatggt 2580

atggagatca gaccacagag acatgatgaa aagaccctcg tgcagtcaca agtgaatgct 2640

tataatgctg atatgattga cccttttcag ttgggccttc tggtcgtgtt cttggccacc 2700

caggaggtcc ttcgcaagag gtggacagcc aagatcagca tgccagctat actgattgct 2760

ctgctagtcc tggtgtttgg gggcattact tacactgatg tgttacgcta tgtcatcttg 2820

gtgggggcag ctttcgcaga atctaattcg ggaggagacg tggtacactt ggcgctcatg 2880

gcgaccttca agatacaacc agtgtttatg gtggcatcgt ttctcaaagc gagatggacc 2940

aaccaggaga acatcttgtt gatgttggcg gctgttttct ttcaaatggc ttatcacgat 3000

gcccgccaaa ttctgctctg ggagatccct gatgtgttga attcactggc ggtagcttgg 3060

atgatactga gagccataac attcacaacg acatcaaacg tggttgttcc gctgctagcc 3120

ctgctaacac ccgggctgag atgcttgaat ctggatgtgt acaggatact gctgttgatg 3180

gtcggaatag gcagcttgat cagggagaag aggagtgcag ctgcaaaaaa gaaaggagca 3240

agtctgctat gcttggctct agcctcaaca ggacttttca accccatgat ccttgctgct 3300

ggactgatta catgtgatcc caaccgtaaa cgcggatggc ccgcaactga agtgatgaca 3360

gctgtcggcc tgatgtttgc catcgtcgga gggctggcag agcttgacat tgactccatg 3420

gccattccaa tgactatcgc ggggctcatg tttgctgctt tcgtgatttc tgggaaatca 3480

acagatatgt ggattgagag aacggcggac atttcctggg aaagtgatgc agaaattaca 3540

ggctcgagcg aaagagttga tgtgcggctt gatgatgatg gaaacttcca gctcatgaat 3600

gatccaggag caccttggaa gatatggatg ctcagaatgg tctgtctcgc gattagtgcg 3660

tacaccccct gggcaatctt gccctcagta gttggatttt ggataactct ccaatacaca 3720

aagagaggag gcgtgttgtg ggacactccc tcaccaaagg agtacaaaaa gggggacacg 3780

accaccggcg tctacaggat catgactcgt gggctgctcg gcagttatca agcaggagcg 3840

ggcgtgatgg ttgaaggtgt tttccacacc ctttggcata caacaaaagg agccgctttg 3900

atgagcggag agggccgcct ggacccatac tggggcagtg tcaaggagga tcgactttgt 3960

tacggaggac cctggaaatt gcagcacaag tggaacgggc aggatgaggt gcagatgatt 4020

gtggtggaac ctggcaagaa cgttaagaac gtccagacga aaccaggggt gttcaaaaca 4080

cctgaaggag aaatcggggc cgtgactttg gacttcccca ctggaacatc aggctcacca 4140

atagtggaca aaaacggtga tgtgattggg ctttatggca atggagtcat aatgcccaac 4200

ggctcataca taagcgcgat agtgcagggt gaaaggatgg atgagccaat cccagccgga 4260

ttcgaacctg agatgctgag gaaaaaacag atcactgtac tggatctcca tcccggcgcc 4320

ggtaaaacaa ggaggattct gccacagatc atcaaagagg ccataaacag aagactgaga 4380

acagccgtgc tagcaccaac cagggttgtg gctgctgaga tggctgaagc actgagagga 4440

ctgcccatcc ggtaccagac atccgcagtg cccagagaac ataatggaaa tgagattgtt 4500

gatgtcatgt gtcatgctac cctcacccac aggctgatgt ctcctcacag ggtgccgaac 4560

tacaacctgt tcgtgatgga tgaggctcat ttcaccgacc cagctagcat tgcagcaaga 4620

ggttacattt ccacaaaggt cgagctaggg gaggcggcgg caatattcat gacagccacc 4680

ccaccaggca cttcagatcc attcccagag tccaattcac caatttccga cttacagact 4740

gagatcccgg atcgagcttg gaactctgga tacgaatgga tcacagaata caccgggaag 4800

acggtttggt ttgtgcctag tgtcaagatg gggaatgaga ttgccctttg cctacaacgt 4860

gctggaaaga aagtagtcca attgaacaga aagtcgtacg agacggagta cccaaaatgt 4920

aagaacgatg attgggactt tgttatcaca acagacatat ctgaaatggg ggctaacttt 4980

aaggcgagca gggtgattga cagccggaag agtgtgaaac caaccatcat aacagaagga 5040

gaagggagag tgatcctggg agaaccatct gcagtgacag cagctagtgc cgcccagaga 5100

cgtggacgta tcggtagaaa tccgtcgcaa gttggtgatg agtactgtta tggggggcac 5160

acgaatgaag acgactcgaa cttcgcccat tggactgagg cacgaatcat gctggacaac 5220

atcaacatgc caaacggact gatcgctcaa ttctaccaac cagagcgtga gaaggtatat 5280

accatggatg gggaataccg gctcagagga gaagagagaa aaaactttct ggaactgttg 5340

aggactgcag atctgccagt ttggctggct tacaaggttg cagcggctgg agtgtcatac 5400

cacgaccgga ggtggtgctt tgatggtcct aggacaaaca caattttaga agacaacaac 5460

gaagtggaag tcatcacgaa gcttggtgaa aggaagattc tgaggccgcg ctggattgac 5520

gccagggtgt actcggatca ccaggcacta aaggcgttca aggacttcgc ctcgggaaaa 5580

cgttctcaga tagggctcat tgaggttctg ggaaagatgc ctgagcactt catggggaag 5640

acatgggaag cacttgacac catgtacgtt gtggccactg cagagaaagg aggaagagct 5700

cacagaatgg ccctggagga actgccagat gctcttcaga caattgcctt gattgcctta 5760

ttgagtgtga tgaccatggg agtattcttc ctcctcatgc agcggaaggg cattggaaag 5820

ataggtttgg gaggcgctgt cttgggagtc gcgacctttt tctgttggat ggctgaagtt 5880

ccaggaacga agatcgccgg aatgttgctg ctctcccttc tcttgatgat tgtgctaatt 5940

cctgagccag agaagcaacg ttcgcagaca gacaaccagc tagccgtgtt cctgatttgt 6000

gtcatgaccc ttgtgagcgc agtggcagcc aacgagatgg gttggctaga taagaccaag 6060

agtgacataa gcagtttgtt tgggcaaaga attgaggtca aggagaattt cagcatggga 6120

gagtttcttt tggacttgag gccggcaaca gcctggtcac tgtacgctgt gacaacagcg 6180

gtcctcactc cactgctaaa gcatttgatc acgtcagatt acatcaacac ctcattgacc 6240

tcaataaacg ttcaggcaag tgcactattc acactcgcgc gaggcttccc cttcgtcgat 6300

gttggagtgt cggctctcct gctagcagcc ggatgctggg gacaagtcac cctcaccgtt 6360

acggtaacag cggcaacact ccttttttgc cactatgcct acatggttcc cggttggcaa 6420

gctgaggcaa tgcgctcagc ccagcggcgg acagcggccg gaatcatgaa gaacgctgta 6480

gtggatggca tcgtggccac ggacgtccca gaattagagc gcaccacacc catcatgcag 6540

aagaaagttg gacagatcat gctgatcttg gtgtctctag ctgcagtagt agtgaacccg 6600

tctgtgaaga cagtacgaga agccggaatt ttgatcacgg ccgcagcggt gacgctttgg 6660

gagaatggag caagctctgt ttggaacgca acaactgcca tcggactctg ccacatcatg 6720

cgtgggggtt ggttgtcatg tctatccata acatggacac tcataaagaa catggaaaaa 6780

ccaggactaa aaagaggtgg ggcaaaagga cgcaccttgg gagaggtttg gaaagaaaga 6840

ctcaaccaga tgacaaaaga agagttcact aggtaccgca aagaggccat catcgaagtc 6900

gatcgctcag cagcaaaaca cgccaggaaa gaaggcaatg ccactggagg gcatccagtc 6960

tctaggggca cagcaaaact gagatggctg gtcgaacgga ggtttctcga accggtcgga 7020

aaagtgattg accttggatg tggaagaggc ggttggtgtt actatatggc aacccaaaaa 7080

agagtccaag aagtcagagg gtacacaaag ggcggtcccg gacatgaaga gccccaacta 7140

gtgcaaagtt atggatggaa cattgtcacc atgaagagtg gagtggatgt gttctacaga 7200

ccttctgagt gttgtgacac cctcctttgt gacatcggag agtcctcgtc aagtgctgag 7260

gttgaagagc ataggacgat tcgggtcctt gaaatggttg aggactggct gcaccgaggg 7320

ccaagggaat tttgcgtgaa ggtgctctgc ccctacatgc cgaaagtcat agagaagatg 7380

gagctgctcc aacgccggta tgggggggga ctggtcagaa acccactctc acggaattcc 7440

acgcacgaga tgtattgggt gagtcgagct tcaggcaatg tggtacattc agtgaatatg 7500

accagccagg tgctcctagg aagaatggaa aaaaggacct ggaagggacc ccaatacgag 7560

gaagatgtaa acttgggaag tggaaccagg gcggtgggaa aacccctgct caactcagac 7620

accagtaaaa tcaagaacag gattgaacga ctcaggcgtg agtacagttc gacgtggcac 7680

cacgatgaga accacccata tagaacctgg aactatcacg gcagttatga tgtgaagccc 7740

acaggctccg ccagttcgct ggtcaatgga gtggtcaggc tcctctcaaa accatgggac 7800

accatcacga atgttaccac catggccatg actgacacta ctcccttcgg gcagcagcga 7860

gtgttcaaag agaaggtgga cacgaaagct cctgaaccgc cagaaggagt gaagtacgtg 7920

ctcaacgaga ccaccaactg gttgtgggcg tttttggcca gagaaaaacg tccaaggatg 7980

tgctctcgag aggagttcat aagaaaggtc aacagcaatg cagctttggg tgccatgttt 8040

gaagagcaga atcaatggag gagcgccaga gaggcagttg aagatccaaa attttgggag 8100

atggtggatg aggagcgcga ggcacatctg cggggggaat gtcacacttg catttacaac 8160

atgatgggaa agagagagaa aaaacccgga gagttcggaa aggccaaggg aagcagagcc 8220

atttggttca tgtggcttgg agctcgcttt ctggagttcg aggctctggg ttttctcaat 8280

gaagaccact ggcttggaag aaagaactca ggaggaggtg tcgagggctt gggcctccaa 8340

aaactgggtt acatcctgcg tgaagttggc acccggcctg ggggcaagat ctatgctgat 8400

gacacagctg gctgggacac ccgcatcacg agagctgact tggaaaatga agctaaggtg 8460

cttgagctgc ttgatgggga acatcggcgt cttgccaggg ccatcattga gctcacctat 8520

cgtcacaaag ttgtgaaagt gatgcgcccg gctgctgatg gaagaaccgt catggatgtt 8580

atctccagag aagatcagag ggggagtgga caagttgtca cctacgccct aaacactttc 8640

accaacctgg ccgtccagct ggtgaggatg atggaagggg aaggagtgat tggcccagat 8700

gatgtggaga aactcacaaa agggaaagga cccaaagtca ggacctggct gtttgagaat 8760

ggggaagaaa gactcagccg catggctgtc agtggagatg actgtgtggt aaagcccctg 8820

gacgatcgct ttgccacctc gctccacttc ctcaatgcta tgtcaaaggt tcgcaaagac 8880

atccaagagt ggaaaccgtc aactggatgg tatgattggc agcaggttcc attttgctca 8940

aaccatttca ctgaattgat catgaaagat ggaagaacac tggtggttcc atgccgagga 9000

caggatgaat tggtaggcag agctcgcata tctccagggg ccggatggaa cgtccgcgac 9060

actgcttgtc tggctaagtc ttatgcccag atgtggctgc ttctgtactt ccacagaaga 9120

gacctgcggc tcatggccaa cgccatttgc tccgctgtcc ctgtgaattg ggtccctacc 9180

ggaagaacca cgtggtccat ccatgcagga ggagagtgga tgacaacaga ggacatgttg 9240

gaggtctgga accgtgtttg gatagaggag aatgaatgga tggaagacaa aaccccagtg 9300

gagaaatgga gtgacgtccc atattcagga aaacgagagg acatctggtg tggcagcctg 9360

attggcacaa gagcccgagc cacgtgggca gaaaacatcc aggtggctat caaccaagtc 9420

agagcaatca tcggagatga gaagtatgtg gattacatga gttcactaaa gagatatgaa 9480

gacacaactt tggttgagga cacagtactg tagatattta atcaattgta aatagacaat 9540

ataagtatgc ataaaagtgt agttttatag tagtatttag tggtgttagt gtaaatagtt 9600

aagaaaattt tgaggagaaa gtcaggccgg gaagttcccg ccaccggaag ttgagtagac 9660

ggtgctgcct gcgactcaac cccaggagga ctgggtgaac aaagccgcga agtgatccat 9720

gtaagccttc agaaccgtct cggaaggagg accccacatg ttgtaacttc aaagcccaat 9780

gtcagaccac gctacggcgt gctactctgc ggagagtgca gtctgcgata gtgccccagg 9840

aggactgggt taacaaaggc aaaccaacgc cccacgcggc cctagccccg gtaatggtgt 9900

taaccagggc gaaaggacta gaggttagag gagaccccgc ggtttaaagt gcacggccca 9960

gcctggctga agctgtaggt caggggaagg actagaggtt agtggagacc ccgtgccaca 10020

aaacaccaca acaaaacagc atattgacac ctgggataga ctaggagatc ttctgctctg 10080

cacaaccagc cacacggcac agtgcgccga caatggtggc tggtggtgcg agaacacagg 10140

atctctaga 10149

<210> 2

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

cgcggatcca ccatggatag gtccatagct ctcacg 36

<210> 3

<211> 58

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

ccggaattct taagcgtaat ctggaacatc gtatgggtaa gcattcactt gtgactgc 58

<210> 4

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

caacctggca gaggtccgca gttattgcta ttt 33

<210> 5

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gtggtggtgc gcccagtgtc agcgtgcacg t 31

<210> 6

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ctgacactgg gcgcaccacc acagagagcg g 31

<210> 7

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

acatgcatgc tgatcttggc tgtccacctc 30

Claims (4)

1. A preparation method of replication-defective West Nile virus is obtained by subculturing NS 1-deleted West Nile virus on Vero/WNV-NS1, wherein the preservation number of the Vero/WNV-NS1 is as follows: CCTCC NO: C2019113.

2. the method of claim 1, wherein the NS 1-deleted West Nile virus is rescued from the infectious clone shown in SEQ ID NO. 1.

3. Use of the process of claim 1 for the preparation of a west nile virus vaccine.

4. The use of the method of claim 1 for the preparation of a reagent for studying west nile virus.

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