Highly immature duck tembusu virus and culture method and application thereof

Abstract

The application discloses a highly immature duck Tembusu virus, a culture method and application thereof, and belongs to the technical field of biotechnology.According to the method for culturing the highly immature duck Tembusu virus based on the LOVO cell line provided in the application, the duck Tembusu virus obtained by culture loses pathogenicity, the application provides a highly potential candidate strain for developing safe and effective duck Tembusu virus vaccines, opens up a new way for safely producing high-quality diagnostic antigens and vaccine antigens, and significantly reduces the biosafety risk of related research.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a highly immature duck Tembusat virus and a culture method and application thereof. BACKGROUND

[0002] Tembusat virus belongs to the same family as DENV, YFV, JEV and ZIKAV, and is a non-segmented, single-stranded RNA virus with an envelope, with a diameter of about 50 nm. The TMUV particle is spherical, including C, prM / M and E three structural proteins and seven non-structural proteins, and has a wide range of natural hosts, including mosquitoes, chickens, ducks, geese, pigeons and sparrows. In 2010, TMUV was introduced into China, and almost all breeds of ducks can be infected with Tembusat virus, among which muscovy ducks are most susceptible to the virus, and the pathogenicity to muscovy ducks is the strongest. After infection, laying hens mainly show reduced egg production and ovarian hemorrhage, and young ducks show clinical symptoms such as neurological symptoms and diarrhea, which has caused great losses to the duck industry in China. Therefore, research and prevention and control work on duck Tembusat virus are very important, and at present, the prevention and control of TMUV mainly relies on vaccination and early diagnosis.

[0003] Most of the existing virus culture methods aim to obtain highly active and infective viruses. For the culture of TMUV, especially the preparation of vaccine antigens, primary DEF cells, BHK-21 or Vero cells are often used, and the virus particles prepared are mainly infectious viruses, which have a certain risk of residual or reverse virulence when preparing vaccines. The production of diagnostic reagents requires a large amount of virus antigens, and the inactivation or attenuation treatment of highly infectious viruses increases the production cost and safety protection cost. In the existing technical scheme, there is a lack of a culture method that can directly, stably and efficiently obtain duck Tembusat virus strains with extremely low infectivity or even no infectivity, while retaining complete or key antigen structures. Therefore, it is of great significance to develop a method for safely culturing TMUV without infectivity to promote the prevention and control of Tembusat virus. SUMMARY

[0004] In view of the above prior art, the present application aims to provide a highly immature duck Tembusat virus and a culture method and application thereof. The duck Tembusat virus cultured by the method provided by the present application has very poor infectivity and basically no infectivity, and can be used in the fields of vaccine development and virus property research.

[0005] To achieve the above-mentioned purpose, the present application adopts the following technical scheme:

[0006] The present application provides a method for culturing a highly immature duck Tembusat virus, comprising the following steps:

[0007] After the LOVO cells are resuscitated and subcultured, 105 TCID 50 TMUV virus, 1-1.5h of sensitization, addition of cell maintenance liquid, 3-5d of culture in 37℃ incubator, collection of virus liquid when 78%-80% of cytopathic effect appears, and high immature duck Tembusat virus is obtained by culture.

[0008] The term "highly immature duck Tembusat virus" mentioned in the present application refers to a form or state of TMUV. The highly immature TMUV virion (imTMUV) has E protein closely combined with prM protein to form heterodimer (E / prM), three heterodimers are arranged to form a spike structure, and assembled into an immature virus particle of about 600Å. The highly immature duck Tembusat virus loses most of the infection ability, loses pathogenicity, and does not affect the key antigen structure.

[0009] In the mature virus particle, the prM on the surface of the virus is cut by furin to remove the N-terminal 91 amino acid pro-peptide pr, leaving the C-terminal 75 residues (mature M protein). At this time, the virus E / prM heterodimer is eliminated, the E protein is rearranged to form E protein homodimer, and then forms a trimer, which is laid on the surface of the virus envelope to expose the functional fusion peptide and the virus receptor binding region. At this time, the virus particle matures, and the diameter is about 500Å.

[0010] Preferably, the cell maintenance liquid is Ham's F-12K liquid medium containing 2% FBS.

[0011] The virus liquid preservation method of the highly immature duck Tembusat virus is: after freezing and thawing once at -80℃, the device is stored at -80℃.

[0012] The key antigen structure is the E / prM structural protein.

[0013] Furin can cleave the precursors of a variety of proteins, including receptors, hormones, viral envelope glycoproteins, growth factors and bacterial exotoxins. The cleavage of premembrin protein (prM) is essential for the production of infectious virions, and Furin plays a crucial role in the cleavage of prM. The maturation of flaviviruses occurs during transport through the secretory pathway, and the viral envelope protein exposes a cleavage site recognized by Furin on prM at low pH, and the prM protein is cleaved into small M protein and pr peptide. When the virus particles are released into the extracellular environment, the virus enters the neutral pH extracellular environment, at which time the pr peptide dissociates from the virion, and the infectious virus containing E and M is produced. Under normal circumstances, only mature virus particles are infectious, and immature particles are not infectious. The prM protein of TMUV has a Furin cleavage site, which can be cleaved into mature virions in cells with Furin, and the Furin activity of LOVO cells is low, which cannot effectively cut the prM protein, and produces non-infectious virus particles with high prM content.

[0014] In a second aspect of the present application, a highly immature duck Tembusu virus is provided, which is prepared by the above method.

[0015] In a third aspect of the present application, the above highly immature duck Tembusu virus is applied in (1) or (2) as follows:

[0016] (1) preparation of a vaccine product for preventing and / or treating duck Tembusu virus;

[0017] (2) preparation of a detection product for preventing duck Tembusu virus.

[0018] Preferably, the vaccine product is any one of inactivated vaccine, attenuated live vaccine and subunit vaccine.

[0019] The detection product is an early diagnosis reagent or early diagnosis kit for preventing duck Tembusu virus.

[0020] The present application has the following beneficial effects:

[0021] The present application provides a method for proliferating and culturing highly immature duck Tembusu virus based on LOVO cell line, and the duck Tembusu virus obtained by using the method provided by the present application has complete virus structure and loses pathogenicity. The present application provides a highly potential candidate strain for developing safe and effective duck Tembusu virus vaccine, opens up a new way for safely producing high-quality diagnostic antigens and vaccine antigens, and significantly reduces the biosafety risk of related research. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1Western-blot identification of TANABUVIR proteins after TMUV infection of LOVO cells.

[0023] Figure 2 IFA identification of TMUV infection of LOVO cells.

[0024] Figure 3 TANABUVIR plaque staining results; wherein Figure 3 A is C6 / 36 amplified TMUV at 10 4 dilution, B is LOVO amplified TMUV at 2 dilution. Figure 3 A is C6 / 36 amplified TMUV at 10

[0025] Figure 4 TMUV gene detection results amplified by different cells; - represents negative control, + represents plasmid positive control.

[0026] Figure 5 TMUV identification results of immature TMUV amplified by LOVO using furin enzyme digestion.

[0027] Figure 6 Infectivity verification of immature TMUV on BHK; wherein, Figure 6 A is LOVO amplified TMUV infection of BHK cells green fluorescence results, Figure 6 B is the corresponding Hoechst cell nucleus staining results. DETAILED DESCRIPTION

[0028] It should be noted that the following detailed description is illustrative only, and is intended to provide further description of the present application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0029] The specific embodiments of the present application will be further described in the following examples. The following detailed description is exemplary in nature and is intended to provide further description of the present application, rather than limit the scope thereof.

[0030] TANABUVIR strain TMUV-SDDZ (MH764606) used in the examples, anti-E monoclonal antibody, NS5 monoclonal antibody and anti-prM (pr) mouse monoclonal antibody, BHK-21 cells and C6 / 36 cells are described in the following documents:

[0031] [1] Siming Zhu, Yi Tang & Youxiang Diao. (2023). Development and biochemical characteristics of a monoclonal antibody against prM protein of Tembusu virus.. Poultry science, 102(12), 103065-103065.

[0032] [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.

[0033] All were preserved by the Avian Disease Laboratory of Shandong Agricultural University, and the public can obtain them from here to reproduce this experiment. LOVO cells were purchased from Wuhan Punsai Life Science and Technology Co., Ltd.

[0034] The following examples were used in the flanking protease purchased from New England Biolabs (NEB) company, HRP labeled goat anti-mouse IgG (H+L), FITC labeled goat anti-mouse IgG (H+L) purchased from Wuhan Sanying Biotechnology Co., Ltd.; BCA protein assay kit, ECL developing kit, TMB developing solution purchased from Suzhou Xinsaimai Biological Technology Co., Ltd.; Fetal bovine serum (FBS), penicillin / streptomycin mixture (P / S 100x), Ham's F-12K cell culture medium, DMDM cell culture medium, phosphate buffered saline (PBS) purchased from Wuhan Punsai Life Science and Technology Co., Ltd.

[0035] Example 1: Highly immature TMUV virus (imTMUV) culture based on LOVO cells

[0036] Cell recovery: The working cell bank LOVO cells derived from ATCC were recovered and passaged, using Ham's F-12K medium supplemented with 10% FBS and 1% P / S, the culture environment was 37°C, 5% CO2, and the cells were passaged when they grew to a single layer.

[0037] Cell passage: wash the cells once with PBS, add 0.25% trypsin to digest the cells, and after the cells appear to be slightly rounded and cracks appear between the cells, discard the trypsin, add the culture medium (10% FBS, Ham's F-12K), blow the cells to disperse them, and pass the cells at a ratio of 1:3.

[0038] Virus amplification: discard the culture medium, wash the cells once with PBS, inoculate 10 5 TCID 50 Tembusu virus SDDZ strain, 1h, add cell maintenance fluid (2% FBS, Ham's F-12K), and place in a 37°C incubator for 3-5 days.

[0039] Harvesting: when about 80% of the cells show cytopathic effect, collect the virus solution, freeze-thaw it once at -80°C, and store it at -80°C.

[0040] Example 2: TMUV virus culture based on C6 / 36 cells (or BHK-21 cells)

[0041] Cell recovery: recover C6 / 36 (or BHK-21 cells) from liquid nitrogen, use DMEM medium with 10% FBS and 1% P / S, and culture in an environment of 30°C (BHK-21 cells are cultured in an environment of 37°C), 5% CO2, and pass the cells when they form a monolayer;

[0042] Cell passage: wash the cells once with PBS, add 0.25% trypsin to digest the cells, and after the cells appear to be slightly rounded and cracks appear between the cells, discard the trypsin, add the culture medium (10% FBS, DMEM), blow the cells to disperse them, and pass the cells at a ratio of 1:3.

[0043] Virus inoculation: discard the culture medium, wash the cells once with PBS, inoculate 10 4 TCID 50 Tembusu virus SDDZ strain, 1h, add cell maintenance fluid (2% FBS, DMEM), C6 / 36 is placed in a 30°C incubator for 4-6 days (or BHK-21 cells are placed in a 37°C incubator for 3-5 days).

[0044] Harvesting: when about 80% of the cells show cytopathic effect, collect the virus solution of the Tembusu virus SDDZ strain, freeze-thaw it once at -80°C, and store it at -80°C.

[0045] Example 3: TMUV infection of LOVO cells

[0046] LOVO cells are recovered and passaged according to the method described in Example 1.

[0047] LOVO cells were subcultured in 12-well cell culture plates, and when the degree of cell fusion was 70-90%, 1 MOI of the tamsui virus SDDZ strain virus liquid collected in Example 2 was inoculated, and at 12h, 24h, 36h, and 48h, at least 10 6 cells were collected at each time for Western Blot to detect TMUV viral proteins Figure 1 ; and after 48h of culture, the cells were fixed using cell fixation solution (4% paraformaldehyde), and indirect immunofluorescence detection was performed using prM antibody as the primary antibody Figure 2 .

[0048] Figure 1 The detection results showed that TMUV can infect LOVO cells and proliferate in them. Figure 2 The detection results showed that tamsui virus SDDZ strain viral proteins can be detected in LOVO cells.

[0049] Example 4: Virus infection plaque and virus titer detection

[0050] Plaque staining test is a classic method for detecting virus infectivity and quantifying virus titer. The infection effect is judged by observing the plaques (cell lesion areas caused by virus infection, not colored) formed by viruses on a monolayer of cells. Each plaque represents a virus particle with infectivity. If the virus infectivity is strong, a large number of plaques will be formed. If the infectivity is weak, there will be very few plaques or even no plaques, which directly reflects the infection effect of the virus. The specific operation is as follows:

[0051] 1. Experimental method

[0052] Virus liquid preparation: The virus liquid harvested in Examples 1 and 2 was centrifuged at 5000 rpm / min for 10 min, and the supernatant was collected and filtered through a sterile 0.22 μm filter for standby use.

[0053] Preparation of virus gradient dilution liquid: Label 6 sterile centrifuge tubes for preparing virus dilution liquid. The above virus liquid was diluted by 10 times gradient using the corresponding culture medium (the virus liquid harvested in Example 1 was diluted by 10 times gradient using Ham's F-12K culture medium added with 10% FBS and 1% P / S, and the virus liquid harvested in Example 2 was diluted by 10 times gradient using DMEM culture medium added with 10% FBS and 1% P / S) for standby use.

[0054] Cell inoculation: C6 / 36 cells were subcultured, and 1×10 6 cells per milliliter of culture medium were inoculated into a 6-well cell plate, and after the cells were cultured to a monolayer, virus inoculation was performed.

[0055] Preparation of agarose: 2% low-melting agarose was treated with high temperature and high pressure, and mixed with DMEM medium containing 2% FBS and 1% P / S at a ratio of 1:1 to prepare agarose culture medium. The agarose culture medium was placed in a 42°C water bath or incubator for standby.

[0056] Infection: The diluted C6 / 36, LOVO amplified TMUV liquid was inoculated into the 6-well cell plate with C6 / 36 cells, 1 ml per well, and incubated for 1 hour. The cells were washed once with PBS, 2 mL of agarose culture medium was added to each well, and the plate was incubated at room temperature for 20 minutes. After the agarose was fully solidified, the plate was inverted and incubated at 30°C for 5-7 days. The plate was observed daily.

[0057] Culture: The 6-well culture plate was placed upside down in a 30°C incubator for 5-7 days, and observed daily.

[0058] Plaque observation and counting: 1 mL of 0.05% neutral red staining (diluted with PBS) was added to each well, and incubated at room temperature for 2-3 hours. The plate was washed multiple times with PBS until no excess dye was present.

[0059] The number of plaques was counted under a microscope, and the virus titer was calculated according to the formula.

[0060]

[0061] 2. Experimental results

[0062] Figure 3 Table A shows the results of C6 / 36 amplified TMUV at a dilution of 10 4 Table B shows the results of LOVO cell amplified TMUV at a dilution of 2. Figure 3

[0063] 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, which could reach more than 10 6 PFU / mL; most of the TMUV cultured in LOVO cells were immature particles, with poor infection ability, and the titer could only reach 10 2 PFU / mL.

[0064] Example 5: Positive detection of TMUV virus E gene

[0065] ​Positive identification was performed on the virus liquid harvested in Examples 1 and 2. 400ul of the virus liquid was taken and placed in a 2ml centrifuge tube, 800ul of Trizol was added, homogenized thoroughly, and allowed to stand at room temperature for 10 minutes; 400ul of chloroform was added, shaken for 15 seconds, and allowed to stand for 10 minutes; centrifuged at 12,000g at 4°C for 15 minutes, 500ul of supernatant was taken to an RNase-free 1.5ml centrifuge tube, an equal volume of cold isopropanol was added, shaken for 15 seconds, and allowed to stand for 10 minutes; centrifuged at 12,000g at 4°C for 10 minutes, the supernatant was discarded; 1ml of cold 75% alcohol was added to wash the RNA precipitate, shaken for 15 seconds, and centrifuged at 8000g at 4°C for 5 minutes; after centrifugation, the supernatant was discarded, the RNA precipitate was air-dried, and RNase-free water was added to dissolve the RNA.

[0066] The RNA was reverse transcribed into cDNA, and the reverse transcription system was 20ul: dNTP Mix 4ul, Primer Mix 2ul, 5xRT Buffer 4ul, DTT 2ul, HiFiScript 1ul, RNA Template 7ul. The reaction program was: 42°C for 50min, 85°C for 5min. cDNA was used as a template for PCR detection, the detection gene was TMUV E gene, and the length of the amplified product was 255bp. The PCR reaction system was 20ul: 2xTaq PCR Master Mix 10ul, upper and lower primers (indicated by SEQ ID NO:1-SEQ ID NO:2) 1ul each, template 2ul, ddH2O 7ul. The PCR amplification conditions were: 95°C for 10min; 95°C for 30s, 55°C for 1min, 72°C for 90s, for 32 cycles; 72°C for 5min. The PCR product was detected by agarose gel electrophoresis, and the detection results showed that both the TMUV amplified by LOVO cells and the TMUV amplified by C6 / 36 cells had E gene, proving that LOVO cells or C6 / 36 cells could be used to amplify TMUV.

[0067] Upstream primer F (SEQ ID NO:1): 5'-CTGAAATAGCGGAAGCACT-3';

[0068] Downstream primer R (SEQ ID NO:2): 5'-AATCCCATTCTCCACTCTTGC-3'.

[0069] Example 6: Detection of structural proteins and non-structural proteins of TMUV amplified by LOVO cells

[0070] LOVO cells were used to expand imTMUV. After the cells showed obvious lesions, the cell supernatant was collected, the cells were discarded, and the cell debris was removed using a centrifuge. After filtration using a 0.22 um filter, the virus was purified using a Beckman ultracentrifuge. After resuspension of the centrifuged product in PBS, the virus was obtained by re-centrifugation using a sucrose gradient.

[0071] The purified virus particles were subjected to enzyme digestion using furin. The enzyme digestion reaction system was 25 μL: 25 μg virus particles, 20 mM HEPES, 0.01% Triton X-100, 1 mM CaCl2, 0.2 mM β-mercaptoethanol, 1 μL furin. The reaction conditions were 25°C for 6 h.

[0072] The enzyme digestion product was concentrated by ultrafiltration using an ultrafiltration tube with a pore size of 100 KD to remove the pr protein cut off in the enzyme digestion system, and the virus particles in the ultrafiltration tube were recovered. Western identification was performed using pr antibody as the primary antibody. The original virus (LOVO expanded immature TMUV) and the virus after furin enzyme digestion (M) were mixed with protein loading buffer, boiled for 10 min, and subjected to SDS-PAGE electrophoresis. The protein bands on the gel were transferred to an NC membrane, which was blocked with 5% skim milk in TBST at room temperature for 2 h. After washing with TBST for 3 times, 1:1000 diluted pr monoclonal antibody was used as the primary antibody and incubated at 4°C overnight. After washing with TBST for 3 times, 1:10,000 diluted goat anti-mouse IgG-HRP was used as the secondary antibody and treated at room temperature for 1 h. After washing with TBST for 3 times, color development was performed using a supersensitive ECL chemiluminescence kit. The detection results are shown in Figure 5 As shown in the figure, the LOVO cell expanded TMUV had viral structural proteins (E / prM) and non-structural proteins (NS5), and the complete prM protein could be detected.

[0073] Example 7: Detection of the infection effect of LOVO cell expanded TMUV

[0074] Cell inoculation: C6 / 36 cells were passaged, and 1×10 5 The cells were inoculated in a 12-well cell plate at 1×10

[0075] Virus infection: The virus liquid collected in Example 1 was inoculated into BHK cells at 0.4 ml per well. After 1 h of infection, cell maintenance liquid (DMEM medium added with 10% FBS and 1% P / S) was added, and the cells were cultured in a cell culture incubator for 48 h.

[0076] Cell fixation: The culture medium in the 12-well plate was discarded, and PBS was used for washing once. 500 μL of cell fixation liquid (4% paraformaldehyde) was added for room temperature fixation for 15 min.

[0077] Blocking: After the cell fixing solution was discarded, PBS was washed twice, 500 μL of 5% BSA was added for blocking, and the blocking was performed at room temperature for 1 h.

[0078] Incubation of the first antibody: After the blocking was completed, washing was performed, the monoclonal antibody against TMUV-prM protein was added, and the incubation was performed at room temperature for 2 h.

[0079] Incubation of the second antibody: After the first antibody was discarded and washing was performed, the second antibody of the goat anti-mouse labeled with FITC was added, and the incubation was performed at room temperature for 1 h.

[0080] Nucleus staining: After the antibody incubation was completed, 500 μL of DAPI was added to each well for staining of the cell nucleus, and the incubation was performed at room temperature for 3-5 min.

[0081] Fluorescence observation: The fluorescence microscope was used to observe the fluorescence, and the green fluorescence was positive.

[0082] The detection results are shown in Table 1. Figure 6 It is shown that the TMUV amplified by using the LOVO cells cannot detect obvious green fluorescence, and it is indicated that the TMUV virus cultured by the method provided in the application cannot effectively infect the BHK cells.

[0083] The above merely describes the preferred embodiments of the present application and is not used to limit the present application. For those skilled in the art, various modifications and changes can be made to the present application. Any modification and change made within the spirit and principle of the present application shall be included in the protection scope of the present application.

[0084] equivalent replacements, improvements, etc. shall be included in the protection scope of the present application.

Claims

1. A method for culturing highly immature duck Tembusu virus, characterized by, It comprises the following steps: After the LOVO cells are recovered and subcultured, TMUV virus is inoculated, and the cells are allowed to be infected for 1-1.5 hours, then cell maintenance solution is added, and the cells are cultured in a 37℃ incubator for 3-5 days; when 78%-80% of the cells show cytopathic effect, the virus solution is collected, and the highly immature duck Tembusu virus is obtained.

2. The culture method according to claim 1, characterized by, The inoculation amount of the TMUV virus is 10 5 TCID 50 .

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

4. The culturing method according to claim 1, wherein, The virus solution of the highly immature duck Tembusu virus is stored in a -80℃ freezer after being frozen and thawed once and then stored in a -80℃ freezer.

5. The culturing method according to claim 1, wherein The highly immature duck Tembusu virus obtained by the method loses pathogenicity and retains key antigen structures.

Images