Highly immature duck tembusu virus as well as culture method and application thereof

Abstract

The invention discloses a highly immature duck tembusu virus as well as a culture method and application thereof, and belongs to the technical field of biology, and the duck tembusu virus cultured according to a method for proliferation culture of the highly immature duck tembusu virus based on an LOVO cell line loses pathogenicity. The invention provides a potential candidate strain for developing a safe and effective duck tembusu virus vaccine, opens up a new way for safely producing high-quality diagnostic antigens and vaccine antigens, and remarkably reduces the biological safety risk of related researches.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a highly immature duck Tembusu virus and a culture method and application thereof. Background Art

[0002] Tembusu virus, along with DENV, YFV, JEV, and ZIKAV, belongs to the Flaviviridae family and the genus Orthomyxovirus. It is an enveloped, non-segmented, single-stranded, positive-sense RNA virus approximately 50 nm in diameter. TMUV particles are spherical and contain three structural proteins (C, prM / M, and E) and seven nonstructural proteins. They have a wide range of natural hosts, including mosquitoes, chickens, ducks, geese, pigeons, and sparrows. TMUV was introduced to China in 2010. Almost all duck breeds are susceptible to Tembusu virus, with Shelduck being the most susceptible and having the highest pathogenicity in ducks. Infection primarily manifests in laying ducks, with clinical symptoms such as decreased egg production and ovarian hemorrhage, and in ducklings, neurological symptoms and diarrhea, causing significant losses to the Chinese duck industry. Therefore, research and prevention of Tembusu virus in ducks are crucial. Currently, prevention and control of TMUV primarily relies on vaccination and early diagnosis.

[0003] Most existing virus culture methods aim to obtain highly active, infectious viruses. For the cultivation of TMUV, especially for the preparation of vaccine antigens, primary DEF cells, BHK-21 cells, or Vero cells are often used. The viral particles produced are mainly infectious viruses, which carries a certain risk of residual virulence or increased virulence when preparing vaccines. The production of diagnostic reagents requires a large amount of viral antigens, and the inactivation or attenuation of highly infectious viruses increases production costs and safety protection costs. Current technical solutions lack a method for directly, stably, and efficiently obtaining duck Tembusu virus strains that naturally have extremely low or even no infectivity, while retaining intact or key antigenic structures. Therefore, developing a method for safely culturing non-infectious TMUV is of great significance for promoting the prevention and control of Tembusu virus. Summary of the Invention

[0004] In view of the above-mentioned existing technologies, the purpose of the present invention is to provide a highly immature duck Tembusu virus and its cultivation method and application. The duck Tembusu virus cultured by the method provided by the present invention has very poor infectivity and basically has no infection ability, and can be used in vaccine development, virus characteristics research and other fields.

[0005] To achieve the above object, the present invention adopts the following technical solutions: The first aspect of the present invention provides a method for culturing highly immature duck Tembusu virus, comprising the following steps: After LOVO cells were recovered and subcultured, 10 5 TCID 50TMUV virus was infected for 1-1.5 hours, and then cell maintenance medium was added and cultured in a 37°C incubator for 3-5 days. When the cells showed 78%-80% cytopathic effect, the virus liquid was collected and cultured to obtain highly immature duck Tembusu virus.

[0006] The term "highly immature duck Tembusu virus" as used herein refers to a specific form or state of TMUV. Highly immature TMUV virions (imTMUV) consist of a tightly bound E protein and prM protein, forming a heterodimer (E / prM). These three heterodimers form a spike structure, assembling into an immature virus particle approximately 600 Å in diameter. Highly immature duck Tembusu virus has lost most of its infectious capacity and pathogenicity, without altering its key antigenic structure.

[0007] In mature virus particles, prM on the surface of the virus is cleaved by furin, throwing off the N-terminal 91 amino acid propeptide pr, leaving the C-terminal 75 residues (mature M protein). At this time, the viral E / prM heterodimer is eliminated, and the E protein rearranges to form an E protein homodimer, and then forms a trimer, which is flattened on the surface of the viral envelope, exposing the functional fusion peptide and viral receptor binding region. At this time, the virus particles are mature and have a diameter of about 500Å.

[0008] Preferably, the cell maintenance medium is Ham's F-12K liquid culture medium containing 2% FBS.

[0009] The method for preserving the highly immature duck Tembusu virus liquid is as follows: freeze and thaw once at -80°C, and then store the liquid at -80°C in separate devices.

[0010] The key antigen structure is the E / prM structural protein.

[0011] Furin cleaves the precursors of a variety of proteins, including receptors, hormones, viral envelope glycoproteins, growth factors, and bacterial exotoxins. Cleavage of the premembrin protein (prM) is essential for the production of infectious virions, and furin plays a crucial role in prM cleavage. Flavivirus maturation occurs during transport through the secretory pathway. Under low pH conditions, the viral envelope protein exposes a furin-recognized cleavage site on prM, cleaving the prM protein into a small M protein and a pr peptide. Upon release into the extracellular environment, the virus enters a neutral pH environment, where the pr peptide dissociates from the virion, producing infectious virions containing E and M. Under normal circumstances, only mature virions are infectious; immature particles are not. The prM protein of TMUV contains a furin cleavage site, allowing it to be cleaved into mature virions in the presence of furin. However, low furin activity in LOVO cells prevents efficient cleavage of the prM protein, resulting in insusceptible virions with a high prM content.

[0012] In a second aspect, the present invention provides a highly immature duck Tembusu virus, which is prepared by the above method.

[0013] The third aspect of the present invention provides the use of the highly immature duck Tembusu virus in the following (1) or (2): (1) Preparation of vaccine products for the prevention and / or treatment of duck Tembusu virus; (2) Prepare detection products for preventing duck Tembusu virus.

[0014] Preferably, the vaccine product is any one of an inactivated vaccine, a live attenuated vaccine and a subunit vaccine.

[0015] The detection product is an early diagnosis reagent or early diagnosis kit for preventing duck Tembusu virus.

[0016] Beneficial effects of the present invention: The present invention provides a method for proliferating and culturing highly immature duck Tembusu virus based on the LOVO cell line. The duck Tembusu virus cultured using the method provided by the present invention has a complete viral structure and loses pathogenicity. The present invention provides a highly potential candidate strain for the development of a safe and effective duck Tembusu virus vaccine, opens up a new approach for the safe production of high-quality diagnostic antigens and vaccine antigens, and significantly reduces the biosafety risks of related research. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Western-blot identification of Tembusu virus protein after TMUV infection of LOVO cells.

[0018] Figure 2 IFA identification of LOVO cells infected with TMUV.

[0019] Figure 3 This is the result of Tembusu virus plaque staining; Figure 3 A is C6 / 36 amplified TMUV at 10 4 Plaque results under dilution multiples, Figure 3 Middle B shows the plaque results of TMUV amplified by LOVO cells at a dilution factor of 2.

[0020] Figure 4 Results of TMUV gene amplification in different cells; - represents negative control, and + represents plasmid positive control.

[0021] Figure 5 The immature TMUV amplified by LOVO was identified by digestion with furin.

[0022] Figure 6 To verify the infectivity of immature TMUV to BHK; Figure 6 Middle A shows the green fluorescence results of LOVO amplified TMUV infected BHK cells. Figure 6 B in the middle shows the corresponding Hoechst cell nucleus staining results. DETAILED DESCRIPTION

[0023] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0024] The following detailed description is for illustrative purposes only and is intended to provide further explanation of the present invention, rather than to limit the scope of the present invention.

[0025] The Tembusu virus strain TMUV-SDDZ (MH764606), anti-E monoclonal antibody, NS5 monoclonal antibody, and anti-prM (pr) mouse monoclonal antibody, BHK-21 cells, and C6 / 36 cells used in the examples are described in the following literature: [1]Siming Zhu, Yi Tang& Youxiang Diao. (2023). Development and biochemical characteristics of a monoclonal antibody against prM protein ofTembusu virus..Poultry science, 102(12),103065-103065. [2]Siming Zhu,Xin Chen,Dalin He,Meijuan Zhang,Xinhong Man,Yi Tang&Youxiang Diao.(2024).Role of long non-coding RNA DLY6E in regulating TMUV infection..Virus research,343,199350-199350. All are preserved by the Avian Disease Laboratory of Shandong Agricultural University and are available to the public for reproduction of this experiment. LOVO cells were purchased from Wuhan Punosai Life Science Technology Co., Ltd.

[0026] Furin enzyme used in the following examples was purchased from New England Biolabs (NEB), HRP-labeled goat anti-mouse IgG (H+L) and FITC-labeled goat anti-mouse IgG (H+L) were purchased from Wuhan Sanying Biotechnology Co., Ltd.; BCA protein assay kit, ECL color development kit, and TMB color development solution were purchased from Suzhou Xinsaimei Biotechnology Co., Ltd.; fetal bovine serum (FBS), penicillin / streptomycin mixture (P / S100×), Ham's F-12K cell culture medium, DMDM ​​cell culture medium, and phosphate buffered saline (PBS) were purchased from Wuhan Punosai Life Science Technology Co., Ltd.

[0027] Example 1: LOVO cell-based culture of highly immature TMUV virus (imTMUV) Cell recovery: LOVO cells from the ATCC working cell bank were recovered and passaged using Ham's F-12K medium supplemented with 10% FBS and 1% P / S at 37°C and 5% CO2. Cells were passaged when they had grown into a complete monolayer.

[0028] Cell passaging: Wash cells once with PBS, add 0.25% trypsin to digest the cells, discard the trypsin after cracks appear between the cells and they become slightly rounded, add culture medium (10% FBS, Ham's F-12K), pipette to disperse the cells, and pass the cells at a 1:3 ratio.

[0029] Virus amplification: Aspirate the culture medium, wash the cells once with PBS, and inoculate 10 5 TCID 50 Tembusu virus SDDZ strain was infected for 1 hour, and then cell maintenance medium (2% FBS, Ham's F-12K) was added to the cells and cultured in a 37°C incubator for 3-5 days.

[0030] Harvest: When the cells show about 80% cytopathic effect, collect the virus solution, freeze and thaw it once at -80℃, and store it in aliquots at -80℃.

[0031] Example 2: TMUV virus culture based on C6 / 36 cells (or BHK-21 cells) Cell recovery: Recover C6 / 36 (or BHK-21) cells from liquid nitrogen using DMEM supplemented with 10% FBS and 1% P / S at 30°C (37°C for BHK-21 cells) and 5% CO2. Subculture when cells have grown into a confluent monolayer. Cell passaging: Wash the cells once with PBS, add 0.25% trypsin to digest the cells, discard the trypsin after cracks appear between the cells and they become slightly rounded, add culture medium (10% FBS, DMEM), pipette to disperse the cells, and pass the cells at a ratio of 1:3.

[0032] Virus inoculation: Aspirate the culture medium, wash the cells once with PBS, and inoculate 10 4 TCID 50 After infecting the Tembusu virus SDDZ strain for 1 hour, add cell maintenance medium (2% FBS, DMEM) and culture C6 / 36 cells in a 30°C incubator for 4-6 days (or culture BHK-21 cells in a 37°C incubator for 3-5 days).

[0033] Harvest: When the cells showed about 80% cytopathic effect, collect the Tembusu virus SDDZ strain virus liquid, freeze and thaw it once at -80℃, and store it in a separate device at -80℃.

[0034] Example 3: Verification of TMUV infection of LOVO cells LOVO cells were revived and passaged according to the method described in Example 1.

[0035] LOVO cells were subcultured and inoculated into 12-well cell culture plates. When the cell fusion degree was 70-90%, 1 MOI of Tembusu virus SDDZ strain virus solution collected in Example 2 was inoculated. At 12 h, 24 h, 36 h, and 48 h, no less than 10 6 cells, and Western Blot was performed to detect TMUV virus protein ( Figure 1After culturing for 48 h, the cells were fixed with a cell fixative (4% paraformaldehyde) and indirect immunofluorescence detection was performed using prM antibody as the primary antibody ( Figure 2 ).

[0036] Figure 1 The test results showed that TMUV could infect LOVO cells and proliferate in them; Figure 2 The test results showed that the viral protein of Tembusu virus SDDZ strain could be detected in LOVO cells.

[0037] Example 4: Virus-infected plaques and virus titer detection Plaque staining is a classic method for detecting viral infectivity and quantifying viral titers. It determines the infection effect by observing the plaques (cell pathological areas caused by viral infection, not stained) formed by the virus on a monolayer of cells. Each plaque represents an infectious virus particle. If the virus is highly infective, a large number of plaques will be formed; if the virus is weakly infective, the number of plaques will be very small or even absent, which directly reflects the infection effect of the virus. Specific steps: 1. Experimental Methods Preparation of virus solution: The virus solution harvested in Examples 1 and 2 was centrifuged at 5000 rpm / min for 10 min. The supernatant was collected and filtered through a sterile 0.22 μm filter for later use.

[0038] Prepare viral dilutions: Label six sterile centrifuge tubes for preparing viral dilutions. Dilute the viral fluid in a 10-fold gradient using the corresponding culture medium (for the viral fluid harvested in Example 1, use Ham's F-12K medium supplemented with 10% FBS and 1% P / S; for the viral fluid harvested in Example 2, use DMEM medium supplemented with 10% FBS and 1% P / S) and set aside.

[0039] Cell seeding: C6 / 36 cells were passaged and 1×10 6 Cells were seeded into 6-well cell plates per ml of culture medium, and virus inoculation was performed after the cells were cultured to a monolayer.

[0040] Prepare agarose: Heat and pressure treat 2% low-melting-point agarose, mix it with DMEM medium containing 2% FBS and 1% P / S in a 1:1 ratio to prepare agarose medium, and place it in a 42°C water bath or incubator until ready to use.

[0041] Infection: Diluted C6 / 36 and LOVO-amplified TMUV fluid were inoculated into 6-well cell plates with C6 / 36 cells, 1 ml per well, incubated for 1 hour, washed once with PBS, and 2 mL of agarose medium was added to each well. The cells were allowed to stand at room temperature for 20 minutes to allow the agarose to fully solidify.

[0042] Culture: Place the 6-well culture plate upside down at 30°C for 5-7 days and observe daily.

[0043] Plaque observation and counting: Add 1 mL of 0.05% neutral red dye (diluted with PBS) to each well, incubate at room temperature for 2-3 hours, and wash several times with PBS until there is no excess dye.

[0044] The number of plaques was counted under a microscope and the virus titer was calculated according to the formula.

[0045] 2. Experimental Results Figure 3 A is C6 / 36 amplified TMUV at 10 4 Plaque results under dilution multiples, Figure 3 Middle B shows the plaque results of TMUV amplified by LOVO cells at a dilution factor of 2.

[0046] The results of virus infection plaque and virus titer detection showed that TMUV cultured in C6 / 36 cells had infection ability and the virus titer was high, reaching 10 6 PFU / mL or more; most of the TMUVs cultured in LOVO cells are immature particles with poor infection ability, and the titer can only reach 10 2 PFU / mL.

[0047] Example 5: Positive detection of TMUV virus E gene Positive identification was performed on the virus fluids harvested in Examples 1 and 2. 400 μl of the virus fluid was placed in a 2 ml centrifuge tube, 800 μl of Trizol was added, and the mixture was thoroughly homogenized and allowed to stand at room temperature for 10 minutes. 400 μl of chloroform was added, the mixture was shaken for 15 seconds, and allowed to stand for 10 minutes. The mixture was centrifuged at 12,000 g for 15 minutes at 4°C. 500 μl of the supernatant was transferred to an RNase-free 1.5 ml centrifuge tube, an equal volume of cold isopropanol was added, the mixture was shaken for 15 seconds, and allowed to stand for 10 minutes. The mixture was centrifuged at 12,000 g for 10 minutes at 4°C, and the supernatant was discarded. The RNA precipitate was washed with 1 ml of cold 75% alcohol, shaken for 15 seconds, and centrifuged at 8,000 g for 5 minutes at 4°C. After centrifugation, the supernatant was discarded, the RNA precipitate was air-dried, and RNase-free water was added to dissolve the RNA.

[0048] RNA was reverse transcribed into cDNA using a 20 μL reverse transcription system consisting of 4 μL dNTP Mix, 2 μL Primer Mix, 4 μL 5× RT Buffer, 2 μL DTT, 1 μL HiFiScript, and 7 μL RNA template. The reaction was performed at 42°C for 50 min and 85°C for 5 min. PCR detection was performed using the cDNA as a template. The target gene was the E gene of TMUV, and the amplified product was 255 bp. The PCR reaction system (20 μL) consisted of 10 μL 2× Taq PCR Master Mix, 1 μL each of the upstream and downstream primers (shown in SEQ ID NO:1-SEQ ID NO:2), 2 μL template, and 7 μL ddH2O. PCR amplification conditions were: 95°C for 10 min, followed by 32 cycles of 95°C for 30 s, 55°C for 1 min, and 72°C for 90 s, and 72°C for 5 min. The PCR products were tested by agarose gel electrophoresis. The results showed that both TMUV amplified from LOVO cells and TMUV amplified from C6 / 36 cells had the E gene, proving that either LOVO cells or C6 / 36 cells can be used to amplify and produce TMUV.

[0049] Upstream primer F (SEQ ID NO: 1): 5′-CTGAAATAGCGGAAGCACT-3′; Downstream primer R (SEQ ID NO: 2): 5′-AATCCCATTCTCCACTCTTGC-3′.

[0050] Example 6: Detection of structural and non-structural proteins of TMUV amplified by LOVO cells LOVO cells were used to amplify imTMUV. After the cells showed obvious pathological changes, the cell supernatant was collected and discarded. Cell debris was removed by centrifuge and filtered using a 0.22 μm filter. The virus was purified using a Beckman ultracentrifuge. The centrifuged product was resuspended in PBS and re-centrifuged using a sucrose gradient to obtain purified virus.

[0051] Purified viral particles were digested with furin. The digestion reaction mixture (25 μL) consisted of 25 μg viral particles, 20 mM HEPES, 0.01% Triton X-100, 1 mM CaCl2, 0.2 mM β-mercaptoethanol, and 1 μL furin. Reaction conditions: 25°C for 6 h.

[0052] The enzyme digestion product was concentrated by ultrafiltration using an ultrafiltration tube with a pore size of 100KD to remove the cleaved pr protein in the enzyme digestion system and recover the virus particles in the ultrafiltration tube. Western identification was performed using pr antibody as the primary antibody. The original virus (immature TMUV amplified by LOVO) and the virus (M) digested by furin enzyme were mixed with protein loading buffer, boiled for 10 minutes, and subjected to SDS-PAGE electrophoresis. The protein bands on the gel were transferred to the NC membrane and the membrane was blocked with TBST containing 5% skim milk for 2 hours at room temperature. Washed three times with TBST, and then used a 1:1000 diluted pr monoclonal antibody as the primary antibody and incubated at 4°C overnight. After washing three times with TBST, a 1:10,000 diluted goat anti-mouse IgG-HRP was used as the secondary antibody and treated at room temperature for 1 hour. After washing three times with TBST, the ultrasensitive ECL chemiluminescence kit was used for color development. The detection results are as follows. Figure 5 As shown, TMUV amplified by LOVO cells contains viral structural proteins (E / prM) and non-structural proteins (NS5), and the complete prM protein can be detected.

[0053] Example 7: Detection of TMUV infection effect on LOVO cell expansion Cell seeding: C6 / 36 cells were passaged and 1×10 5 Cells were seeded into 12-well cell plates per milliliter of culture medium, and virus inoculation was performed after the cells were cultured to a monolayer.

[0054] Virus infection: The virus solution collected in Example 1 was inoculated into BHK cells at a rate of 0.4 ml per well. After 1 hour of incubation, cell maintenance medium (DMEM supplemented with 10% FBS and 1% P / S) was added and the cells were cultured in a cell incubator for 48 hours.

[0055] Cell fixation: Discard the culture medium in the 12-well plate, wash once with PBS, add 500 μL of cell fixative (4% paraformaldehyde) and fix at room temperature for 15 minutes.

[0056] Blocking: After discarding the cell fixative, wash twice with PBS, add 500 μL 5% BSA for blocking, and block at room temperature for 1 hour.

[0057] Incubation with primary antibody: After blocking, wash and add monoclonal antibody against TMUV-prM protein and incubate at room temperature for 2 hours.

[0058] Incubation with secondary antibody: After discarding the primary antibody and washing, add FITC-labeled goat anti-mouse secondary antibody and incubate at room temperature for 1 hour.

[0059] Nuclear staining: After antibody incubation, add 500 μL of DAPI to each well to stain the cell nuclei and incubate at room temperature for 3-5 minutes.

[0060] Fluorescence observation: Use a fluorescence microscope to observe fluorescence, and green fluorescence is positive.

[0061] Test results such as Figure 6 The results showed that no obvious green fluorescence could be detected when TMUV was amplified using LOVO cells, indicating that the TMUV virus cultured using the method provided by the present invention could not effectively infect BHK cells.

[0062] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification made within the spirit and principles of the present application shall be Equivalent replacements, improvements, etc. should all be included in the protection scope of this application.

Claims

1. A method for culturing highly immature duck Tembusu virus, characterized in that: The steps include: After the LOVO cells were revived and subcultured, they were inoculated with TMUV virus and infecting for 1-1.5 hours. Cell maintenance medium was added and cultured in a 37°C incubator for 3-5 days. When the cells showed 78%-80% cytopathic effect, the virus liquid was collected and cultured to obtain highly immature duck Tembusu virus.

2. The culture method according to claim 1, wherein The inoculum size of the TMUV virus was 10 5 TCID 50 .

3. The culture method according to claim 1, wherein The cell maintenance medium is Ham's F-12K liquid culture medium containing 2% FBS.

4. The culture method according to claim 1, wherein The method for preserving the highly immature duck Tembusu virus liquid is as follows: freeze and thaw once at -80°C, and then store the liquid at -80°C in separate devices.

5. The culture method according to claim 1, wherein The highly immature duck Tembusu virus cultured by the method loses pathogenicity and retains key antigenic structures.

6. A highly immature duck Tembusu virus, characterized in that The highly immature duck Tembusu virus is prepared by the culture method according to claim 1.

7. Use of the highly immature duck Tembusu virus according to claim 6 in the following (1) or (2): (1) Preparation of vaccine products for the prevention and / or treatment of duck Tembusu virus; (2) Prepare detection products for preventing duck Tembusu virus.

8. The use according to claim 7, characterized in that The vaccine product is any one of an inactivated vaccine, a live attenuated vaccine and a subunit vaccine.

9. The use according to claim 7, characterized in that The detection product is an early diagnosis reagent or early diagnosis kit for preventing duck Tembusu virus.

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