GB2589230A - Replication-defective recombinant H9N2 avian influenza virus expressing HA of H5 subtype - Google Patents
Replication-defective recombinant H9N2 avian influenza virus expressing HA of H5 subtype Download PDFInfo
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- GB2589230A GB2589230A GB2019165.6A GB202019165A GB2589230A GB 2589230 A GB2589230 A GB 2589230A GB 202019165 A GB202019165 A GB 202019165A GB 2589230 A GB2589230 A GB 2589230A
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Abstract
A method for preparing a replication-defective recombinant H9N2 avian influenza virus expressing HA of H5N1 subtype, comprising constructing a replication-defective recombinant avian influenza virus steadily expressing surface glycoprotein hemagglutinin (HA) of both H9N2 and H5N1 subtypes and subjecting to replication and packaging in a MDCK cell capable of steadily expressing neuraminidase (NA) of both H9N2 subtype to form a recombinant virus particle. The virus particle can be used to prepare an attenuated vaccine.
Description
Descriptions
Replication-defective Recombinant H9N2 Avian Influenza Virus Expressing HA of H5 Subtype
Technical Field
This invention belongs to the technology research and development field of influenza virus vaccine, in particular to a replication-defective recombinant H9N2 Avian influenza virus expressing HA of H5 subtype; that is, a recombinant influenza virus expressing surface glycoprotein hemagglutinin (HA) of both H9N2 and H5N1 subtypes of Avian influenza virus, and its preparation method and application.
Background Technology
Avian influenza is an infectious disease of the respiratory system and systemic virimia in poultry caused by Avian influenza virus (A1V). H5N1 subtype Avian influenza virus (MV) is a highly pathogenic virus, which is highly infectious and severely harmful to poultry, human beings and other animals. H9N2 subtype is a low pathogenic Avian influenza virus, which widely exists in the world. Its harm is perpetual and difficult to control, in particular, the coinfection leads to high mortality. H9N2 influenza virus is also the internal gene donor of H5N1, H7N9 and other subtypes of influenza viruses, which can generate new recombinant viruses and seriously endanger the development of poultry industry and public health in China. Therefore, the research and prevention of H9N2 and H5N1 influenza viruses should not be ignored. At present, vaccination is still the most important and effective measure to prevent and control Avian influenza. Only inactivated vaccine has been approved for H5N1 and H9N2 Avian influenza viruses, and there is not any live attenuated vaccine that can prevent both H9N2 and H5N1 Avian influenza viruses either. In addition, conventional live attenuated influenza vaccines are mostly cold-adapted strains, and there is a risk of virulence reversion mutation.
Invention Summary
This invention provides a replication-defective recombinant H9N2 Avian influenza virus expressing HA of H5 subtype, its preparation method and application in vaccine preparation.
This invention first provides a replication-defective recombinant H9N2 Avian influenza virus expressing HA of H5 subtype, of which the preparation method is as follows: 1) Insert the Open Reading Frame (ORE) of hemagglutinin HA gene of H5N1 subtype into the packaging signal region at 51end and 3' end of neuraminidase (NA) of H9N2 virus to form the DNA fragment for synthesizing Avian influenza virus NAps-HA-NAps; The described packaging signal region at Send is 203 base pairs at 5' end of
Descriptions
neuraminidase (NA) of H9N2; The described packaging signal region at 3' end is 195 base pairs at 3' end of neuraminidase (NA) of 119N2; Furthermore, the described DNA fragment for synthesizing Avian influenza virus NA-HA-NA ps has a nucleotide sequence of SEQ ID NO:]; 2) Clone the DNA fragment constructed in 1) for synthesizing Avian influenza virus NAp-FIA-NAps into the plasmid to form a recombinant plasmid; The described plasmid is preferably the plasmid pHW2000, 3) Construct MDCK cell lines expressing neuraminidase (NA)gene of H9N2 influenza virus; 4) Transfect the recombinant plasmid constructed in 2) together with the recombinant plasmid of each gene fragment of PB2, PB1, PA, HA, NP, M and NS expressing H9N2 into 293T cell and MDCK cell lines stably expressing neuraminidase (NA) gene of H9N2 influenza virus, and collect culture supernatant of transfected 293T cell and MDCK cell expressing NA.
5) Propagate the rescued recombinant influenza virus in 4) in MDCK cell lines stably expressing neuraminidase (NA) of H9N2 subtype Avian influenza virus.
The replication-defective recombinant H9N2 Avian influenza virus expressing HA of 115 subtype of this invention is used for preparing attenuated vaccine This invention rescues a bivalent live attenuated vaccine strain of replication-defective Avian influenza virus. Without changing the gene stability, the constructed recombinant virus can stably express the surface glycoprotein hemagglutinin (HA) of both H9N2 and 115N1 subtypes of Avian influenza virus. The attenuated virus not only has good gene stability, but also cannot replicate in experimental animals, so it is not pathogenic. At the same time, it can also induce the body to produce strong mucosal immune response and cellular immune response, and maintain strong and lasting immunogenicity. Most importantly, the vaccine candidate strain can produce immune protection against 119N2 and H5N1 subtypes of Avian influenza virus. Therefore, the prevention and control of Avian influenza has great social significance.
Brief Description of Drawings
Figure 1: The technology road map for embodiment of this invention;
Descriptions
Figure 2: Gel electrophoresis map of the DNA sequence for synthesizing NA-HA-NA; Figure 3: Growth curve of A830-HA virus.
Figure 4. The survival rate of experimental animals after challenge with different titers of virus solution of recombinant A830-HA.
Figure 5: The survival rate of experimental animals after immunization with virus solution of recombinant A830-HA.
Detailed Description of the Presently Preferred Embodiments This invention present the development of a replication-defective recombinant Avian influenza virus expressing surface glycoprotein hemagglutinin HA of H9N2 and H5N1 subtypes of Avian influenza virus, and preparation of live attenuated vaccine.
In order to more clearly explain the specific embodiments of this invention or the technical solutions in the prior art, the drawings needed in the description of specific embodiments or prior art will be briefly introduced as follows: Embodiment 1 Construction of DNA fragment and plasmid for synthesizing NAps-1-1A-NAps Avian influenza virus Plasmid pHW-PB2, pHW-PB1, p1-1W-PA, pHW-HA, pHW-NP, pHW-M and pHW-NS that include the Avian influenza virus H9N2 were used. These seven plasmids constituted a reverse genetic system for rescue of H9N2 Avian influenza virus.
1. Construct DNA fragment for synthesizing Avian influenza virus NAps-HA-NApss Synthesize the DNA sequence of Avian influenza virus NA-HA (ORF): Replace the Open Reading Frame (ORF) of the neuraminidase (NA) in Avian influenza virusA/Chicken/Shandong/830/2014 (H9N2) with the Open Reading Frame of hemagglutinin (HA) of A/wildduck/Hunan/021/2005 (H5N1) subtype of Avian influenza virus while retaining the packaging signal region at 5' end (203 base pairs) and 3' end (195 base pairs) of NA to synthesizea new DNA sequence named NAps-HA(ORF) -NAps(as shown in Figure 1) 2. Construct plasmid pHVV-NA-HA Firstly, synthesize DNA sequence NA-HA-NA ps (Genscript), which retained the
Descriptions
packaging signals of 203 nucleotides in the non-coding region of 3' end and 195 nucleotides in the non-coding region of 5' end of NA fragment of Avian influenza virus H9N2. Propagate the target fragment NA(203nt)-ORF(HA)-NA(195nt) by PCR technology (Figure 2).
Forward primer sequence TATTGGTCTCAGGGAGCAAAAGCAGGAGT, backward primer sequence: ATATGGTCTCGTATTAGTAGAAACAAGGAGTTTTTT, digestand analyzeplasmid pHW2000 and PCR product NA-HA-NA ps with BsmB1 and Bsal by restriction enzymerespectively; ligate the above two restriction fragments with T4 DNAligase to cloneplasmid pHW-NA-HA.
The DNA sequence of the newly synthesized target fragment NA(203nt)-ORF(HA)-NA(195n0 was SEQ ID NO:1: Embodiment 2 Rescue recombinant virus Construct cell lines: Construct MDCK cell lines stably expressing surface glycoprotein neuraminidase (NA) of A/Chicken/Shandong/830/2014 (H9N2)influenza virus.
The specific steps are as follows: Screen stably expressing celllines with G418 Before screening, the optimal concentration of G418 for screening MDCK cell was determined to be 500 Rg/mL Preparation of G418: Dissolve lg G418 in ImL 1M HEPES solution, add ultrapure water to 10m1, filter and store it at 4°C for later use.
(1) Reverse transcribe in vitro the RNA fragment of the surface glycoprotein neuraminidase (NA) of H9N2 subtype of Avian influenza virus into cDNA, propagate the NA gene fragment of influenza virus with cDNA as template, and clone it into vector plasmid pD2EGFP-N1. The plasmid pD2EGFP-NA was obtained.
(2) Lay MDCK cells on a 6-well plate and culture them in MEM medium containing 10% Fetal Bovine Serum (FBS) and 1% Penicillin-Streptomycin Solution (PS) in a 37°C incubator. (The culture medium and serum were purchased from Biological Industries). Transfect the cells when their growth density reached 60-70%.
(3) Before transfection, suck out the culture medium from the 6-well cell culture plate and discard it, wash the cells twice with PBS, add fresh opti-MEM medium, and put the culture plate back into the incubator.
Descriptions
(4) Prepare transfection reagent: Solution A: Add 100uL opti-MEM medium into a sterile 1.5 ml centrifuge tube, then add 2ug plasmid pD2EGFP-NA, mixed it gently, and let stand at room temperature for 5 minutes.
Solution B: Add 100 [IL opti-MEM medium into a sterile 1.5mL centrifuge tube, and then add 6-8[IL Lipofectamine2000 (Invitrogen) transfection reagent. After that, slowly add solution A to solution B and let stand at room temperature for 20 minutes.
(5) Take out the 6-well plate, add the mixture in (4) to the wells, shake it and mix well.
(6) After 6 hours of transfection, suck out the culture medium from the 6-well plate and discard it, add fresh NIEM medium containing 10% Fetal Bovine Serum (FBS) and 1% Penicillin-Streptomycin Solution (PS), and culture it for 24 hours.
(7) After 24 hours of transfection, observe the 6-well cell culture plate with a fluorescence microscope. If the expression of obvious green fluorescence in MDCK cells (because there is a gene sequence EGFP expressing green fluorescence on the vector plasmid pD2EGFP-N1), it can be concluded that the plasmid pD2EGFP-NA has been successfully transfected into MDCK cells.
(8) After 24 hours of transfection of the cells, if the expression of green fluorescence is observed under microscope, the culture medium can be replaced by MEM medium containing 10% Fetal Bovine Serum (FBS) and 500ug/mL G418 for cell screening Change culture medium once every 4-5 days until all other cells die. Screening is considered successful if only the positive cloned cells are left.
(9) Culture the successfully screened cells and verify whether they can proliferate stably. If they can proliferate stably, it is proved that MDCK cell lines stably expressing surface glycoprotein neuraminidase (NA) of A/Chicken/Shandong/830/2014 (I-19N2) influenza virus are constructed successfully.
Culture of cell lines: 293T cells were cultured in DA/IFNI (Dulbecco' s Modified Eagle' s Medium) containing 10% Fetal Bovine Serum (FBS) and 1% Penicillin-Streptomycin Solution (PS) in 37°C incubator. The modified MDCK cells (stably expressing neuraminidase (Na)) were cultured in MEM medium containing 10% Fetal Bovine Serum (FBS) and 1% Penicillin-Streptomycin Solution (PS) in 37°C incubator. (The culture medium and serum were purchased from Biological Industries).
Cell lines transfection: Lay 293T cells and modified MDCK cells in a ratio of 1:1 in a 6-well cell culture plate with 7x105 cells in each well, and transfect them when the cell growth density reached 60%-70%. Before transfection, suck out the original
Descriptions
DMEM medium from the 6-well cell culture plate and discard it, wash the cells twice with Phosphate Buffer Saline (PBS), and add 2 mL fresh Opti-MEM medium. Transfect eight plasmids, namely pHW-PB2, pHW-PB1, pHW-PA, pHW-HA, pHW-NP, pHW-NA-HA, pHW-M and pHW-NS, into 293T cells and MDCK cells with the transfection reagent Lipofectamine2000 (1nvitrogen). After six hours of transfection, discard the culture medium in the 6-well cell culture plate and add 2mL fresh Opti-MEM medium; after 24 hours of transfection, add 1 pg/mL of Tosylsufonyl Phenylalanyl Chloromerthyl Ketone-trypsin (TPCK trypsin)into the culture medium. After 48 more hours of culture, collect the cell supernatant.
Embodiment 3 The growth curve of tested recombinant virus MDCK cells stably expressing NA were cultured in _MEM medium containing 10% Fetal Bovine Serum (FBS) and I% Penicillin-Streptomycin Solution (PS) in 37°C incubator. (The medium and serum were purchased from Biological Industries). Lay the modified MDCK cells in a 6-well cell culture plate with 3x105 cells in each well. Propagate the virus when the cell growth density reached 90%.Before virus propagation, wash the modified MDCK cells twice with Phosphate Buffer Saline (PBS), infect the cells with recombinant virus with MoI=0.001, after one hour of adsorption, add 2mL MEN1 medium containing 0.2% Bovine Serum Actin (BSA) and I jtg/mL TPCK, collect the supernatant at 12, 24, 48 and 72 hours after infection respectively and store it at -80°C.
Embodiment 4 Measure the titer of virus by plaque assay The assay was divided into the following two groups: Plaque assay was performed in MDCK cells stably expressing NA of A/Chicken/Shandong/830/2014 (H9N2) influenza virus with wild-type A/Chicken/Shandong/830/2014 (H9N2) influenza virus used in Group 1 and rescued recombinant virus used in Group 2 Plaque assay: (1) Lay MDCK cells stably expressing NA evenly in two 6-well plates with 8x105 cells in each well. Prepare serum-free 2 xDMEM medium, adjust its PH value to 7-7.5 with NAOH solution, and filter it with filter for later use.
Prepare 1.6% low melting point agarose: Weigh 0.18g agarose and dissolve it in 15mL ultrapure water, microwave it for 40s until completely dissolved, and then put it into a 42°C water bath for constant temperature preservation.
(2) Dilute virus: Take eight 1.5mL centrifuge tubes labeled as No.1-8, and add 900 pL serum-free DMEM medium into the centrifuge tubes respectively; then add 100 tit virus solution into tube 1, mix well, take
Descriptions
100p,L, liquid from tube 1 and add it into tube 2, mix well, and dilute to tube 8 in turn by doubling dilution.
(3) Take out the 6-well plate, absorb and discard the culture medium, and wash it with PBS containing calcium and magnesium ions. Add virus solution with dilution concentration of 10-3-10-8into well 1-6 and keep it in 37°C incubator for 1h. During this period, take it out every 15 minutes, shake it gently and mix well to make the virus solution fully absorbed.
(4) Prepare maintenance solution: Add 1200RL 5%BSA, 300 nonessential amino acid NEAA and 37.5 RE TPCK-trypsin storage solution with concentration of lmg/mL at 37° C into 13.5mL serum-free 2xDMED medium, and keep it in a 37° C incubator.
(5) After one hour, take out the 6-well plate, suck out the remaining virus solution in the wells and discard it, and wash it with PBS containing calcium and magnesium ions. Mix 1.6% low melting point agarose and maintenance solution. After mixing, add 4mL of it into each well of the 6-well plate. After solidification, put it in a 37°C incubator for culture.
(6) After 3 days, take out the 6-well plate and discard the gel in the wells. Add 500 iL Coomassie Brilliant blue to each well, let stand at room temperature for 5 minutes, slowly wash off the dye with water, observe and count the plaque number of each well, and make the growth curve of the virus, as shown in Figure 3. The growth curve of A830-HA recombinant virus in the experimental group had the same tendency as that of A830 virus in cells, indicating that recombinant virus A830-11A has a good replication and proliferation ability in MDCK cell lines stably expressing NA.
Embodiment S Animal experiment 1. Safety test: Study the safety of recombinant virus A830-HA in SPF chickens with the survival rate of SPF chickens after challenge as an index.
Divide healthy SPF chickens aged 4-5 weeks into 4 groups randomly with 8 chickens in each group. Observe and calculate the survival rate of SPF chickens in each group every day after intranasal inoculation and challenge with three recombinant influenza viruses with different titers, specifically, 105, 106 and 107PFU, as shown in Figure 4. The survival rate of SPF chickens was not affected by the intranasal challenge of A830-HArecombinant influenza virus with different titers, indicating that the recombinant virus A830-HA is not infectious to the body in the absence of exogenous NA.
2. Immune protection experiment: Study on neutralization test of the replication-defectivebivalent live attenuated vaccine against Avian influenza virusH9N2 and H5N1.
Divide healthy SPF chickens aged 4-5 weeks into 4 groups randomly with 8
S
Descriptions
chickens in each group. The first group was negative control group, which was inoculated with PBS; the second group was positive control group, which was inoculated with 10PFU/mouse A830 virus solution in a transnasal way, and the third and the fourth group were inoculated with 10PFU/mouse and 100PFU/mouse A830-HA recombinant virus solution respectively. On the 29th day after inoculation, the above four groups (Table 1) were challenged with 10xLD50 wild-type A830 virus solution and survival rate was calculated (As shown in Figure 5, the experimental group inoculated with 10 PFU/mouse and 100 PFU/mouse A830-HA recombinant virus had the same weight and survival rate as the positive control group, indicating that the A830-HA recombinant virus had good immune effect on the body).
Table 1 Immune challenge list of mice Group No. Immunity (PFU/mouse) Challenge 1 None 10x LD5o A830 2 10 PFU wide-type A830 10x LD50 A830 3 10 PFU 10x LIDso A830 4 100 PFU 10x LD50 A830 Collect the blood of SPF chickens in the above four groups, separate the serum; carry out hemagglutination inhibition test and virus neutralization test.
The titer of serum antibody against wild-type A830 virus was determined by hemagglutination inhibition test: (1) Place the serum to be tested on ice, melt it for later use. Take V-type 96-well microreaction plate and mark it.
(2) Add 25 1jT.. normal saline into well 1-10 and 50!IL normal saline to well 11 with micropipette.
(3) Suck 25 pi, of the serum to be tested with micropipette, put it into the well 1. Immerse the tip in the liquid and slowly blow and suck for a few times to make the serum to be tested and diluent mix evenly. Then suck 25 pL liquid and add it into well 2. Repeat this dilution step until well 10. Finally, suck out 25 pL liquid and discard it. The dilution ratio of tested serum was 1:2-1:1024. Well 11 was red blood cell control and well 12 was antigen control.
(4) Add 25 tL virus solution containing 4 units into each of well I to 10. Well 11 was red blood cell control well not containing virus solution; well 12 was antigen control well containing virus solution.
(5) Put it on the oscillator to oscillate for 1-2min, then keep it at 37°C for 20min.
( 6) Add 25pL 0.8% red blood cell suspension into each well, put it on a micro oscillator to oscillate for 1-2min, mix well, keep it at 37°C, and determine the result after 15min.
(7) Results determination: Agglutination was shown as that red blood cells laid on the bottom wall of V-tube; unagglutinated red blood cells were shown as
Descriptions
obvious red dots in the V-shaped agglutination pore. Determine the results after tilting the reaction plate by 45 degrees. The inhibition rate was 100% when the red blood cells showed teardrop like flow without agglutinated particles.
( 8) Results calculation: Calculate it by Reed-Muench methods or Karber method.
Table 2: The titer of serum antibody against wild-type A830 virus was determined by hemagglutination inhibition test Group (n) Immunity (PFU/mouse) Challenge 1 None 4+2 2 10 PFU A830 1024+8 3 10 PFU A830-HA 1024±8 4 10 PFU A830-HA 1024+4 According to the hemagglutination titer data in Table 2, the hemagglutination titers in Group 2, 3 and 4 are very high, indicating that the concentration of serum antibody against A/Chicken/Shandong/830/2014 (H9N2) is very high, which indirectly indicates that the recombinant virus A830-HA has a good immune protection effect on FI9N2 subtype of influenza virus infection.
Virus micro neutralization test: ( 1) Lay MDCK cells in the 96-well cell culture plate.
( 2) Keep the serum to be tested at 56°C for inactivation.
(3) Dilute the serum to be tested by doubling dilution, that is, adding 100PFU recombinant A830-HAvirus solution into each tube of serum with different dilution times.
(4) Take out the 96-well cell culture plate for culturing MDCK cells from the incubator, discard the culture medium, and wash it twice with PBS.
(5) Add the prepared mixture of serum with different dilution times and virus as described in (3) into a 96-well cell culture plate, mark it, and culture it in 37°C incubator for 72 hours.
( 6) Detect neutralization titer Table 3: Virus micro neutralization experiment Group (n) Immunity (PHI/mouse) Challenge 1 None 4±2 2 10 PFU 4+2 3 10 PFU 1024+8 4 10 PFU 1024+4 According to the neutralization titer data in Table 3, the neutralization titers of Group 3 and 4 are very high, indicating that the concentration of serum antibody against A/wild duck/Hunan/021/2005 (H5N1) is very high, and recombinant virus
Descriptions
A830-HAhas a good immune protection effect on H5N1 subtype of influenza virus infection.
Claims (9)
- Claims 1. A method for preparing replication-defective recombinant H9N2 Avian influenza virus expressing HA of 115 subtype, characterized in that the described method is as follows: 1) Insert the Open Reading Frame (ORF) of hemagglutinin HA gene of H5N1 subtype into the packaging signal region at 5'end and 3' end of neuraminidase (NA) of H9N2 virus to form the DNA fragment for synthesizing Avian influenza virus NA-HA; 2) Clone the DNA fragment constructed in 1) for synthesizing Avian influenza virus NA-HA into the plasmid to form a recombinant plasmid; 3) Construct MDCK cell lines expressing neuraminidase (NA)gene of H9N2 influenza virus; 4) Transfect the recombinant plasmid constructed in 2) together with the recombinant plasmid of each gene fragment of PB2, PB1, PA, HA, NP, M and NS expressing H9N2 into 293T cell and MDCK cell lines stably expressing neuraminidase (NA) gene of H9N2 influenza virus, and collect culture supernatant of transfected 293T cell and M1DCK cell expressing NA; 5) Propagate the rescued recombinant influenza virus in 4) in MDCK cell lines stably expressing neuraminidase (NA) of H9N2 subtype Avian influenza virus.
- 2. The preparation method described in Claim 1 is characterized in that the described packaging signal region at 5' end in 1) is 203 base pairs at 5' end of neuraminidase (NA) of 119N2;
- 3. The preparation method described in Claim 1 is characterized in that the described packaging signal region at 3' end in 1) is 195 base pairs at 3' end of neuraminidase (NA) of 119N2;
- 4. The preparation method described in Claim 1 is characterized in that the described DNA fragment for synthesizing Avian influenza virus NA-HA has a nucleotide sequence of SEQ ID NO: I;
- 5. The preparation method described in Claim 1 is characterized in that the described plasmid in 2) is the plasmid pHW2000.
- 6. A replication-defective recombinant H9N2 Avian influenza virus expressing HA of H5 subtype, characterized in that the described recombinant H9N2 Avian influenza virus is prepared by the method described in Claim I. Claims
- 7. The application of the recombinant H9N2 Avian influenza virus as described in Claim 6 in the preparation of vaccine.
- 8. The application described in Claim 7 is characterized in that the described vaccine is an attenuated vaccine.
- 9. An attenuated vaccine, characterized in that the antigen used in the described attenuated vaccine contains the recombinant H9N2 Avian influenza virus as described in Claim 6.
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| PCT/CN2018/114270 WO2020052035A1 (en) | 2018-09-14 | 2018-11-07 | Replication-defective recombinant h9n2 avian influenza virus expressing ha of h5 subtype |
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| CN114836390B (en) * | 2022-06-13 | 2023-05-02 | 华南农业大学 | H9N2 subtype avian influenza virus MDCK cell cold-adaptation attenuated live vaccine strain and application thereof |
| CN115998854A (en) * | 2023-01-30 | 2023-04-25 | 南京市第二医院 | Application of non-coding region of influenza virus genes in preparation of mRNA vaccine |
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| CN109136199A (en) | 2019-01-04 |
| CN109136199B (en) | 2020-02-07 |
| GB202019165D0 (en) | 2021-01-20 |
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