Duck tembusu virus attenuated strain and application thereof

Abstract

The application discloses a duck Tembusu virus attenuated strain, and a nucleotide sequence of the duck Tembusu virus attenuated strain is shown as SEQ ID NO:1.The duck Tembusu virus attenuated strain can immunize ducklings and make the ducklings produce effective duck Tembusu virus antibodies, the protection rate of the ducklings reaches 100%, meanwhile, the weight growth of the ducklings is not affected, and there is no virus discharge, death and viremia, and the duck Tembusu virus is not detected in main organs of the ducklings.The duck Tembusu virus attenuated strain can also be used as a live vector to embed H5N6 subtype avian influenza virus antigen epitopes, and further construct a live vector vaccine strain, and the live vector vaccine strain can immunize ducklings and effectively reduce the damage of H5N6 subtype avian influenza virus to the ducklings.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to an attenuated duck Tembusu virus strain and applications thereof. Background Art

[0002] Duck Tembusu virus (DTMUV) infection is an acute, febrile infectious disease of waterfowl, causing decreased egg production, reduced feed intake, and paralysis, with a mortality rate of 5% to 30%. DTMUV can infect a variety of bird species, including ducks, chickens, geese, pigeons, and sparrows, with ducks being the most susceptible. Therefore, DTMUV infection poses a serious threat to the healthy development of the duck industry.

[0003] Currently, the prevention and control of DTMUV relies primarily on inactivated and attenuated vaccines. However, inactivated vaccines can only induce humoral immune responses and have limited immune efficacy. Attenuated vaccines are all produced by passage through duck and chicken embryos or cells, with a long production cycle, making them inadequate for responding to sudden outbreaks. However, genetically engineered live attenuated DTMUV vaccines, developed using reverse genetics, can induce both humoral and cellular immune responses and have a short production cycle, potentially addressing the shortcomings of traditional DTMUV vaccines. Genetically engineered live attenuated vaccines can also be used as live viral vectors, incorporating antigenic proteins or epitopes from other viruses to develop live vector vaccines for disease prevention.

[0004] The H5N6 subtype avian influenza virus is one of the most prevalent avian influenza viruses (AIV) in my country. The mortality rate of poultry infected with this virus is as high as 100%. Furthermore, the H5N6 subtype avian influenza virus can also infect humans, posing a serious threat to the development of the poultry industry and public health safety. Currently, the prevention and control of the H5N6 subtype avian influenza virus mainly relies on inactivated vaccines, but these vaccines can only induce a humoral immune response, not a cellular immune response. Furthermore, there are currently no reports of using the duck Tembusu virus vaccine strain as a live vector to develop an avian influenza vaccine.

[0005] Therefore, there is an urgent need for an attenuated vaccine strain of duck Tembusu virus to make up for the shortcomings of the existing Tembusu vaccine, and to use the attenuated vaccine strain of duck Tembusu virus to develop a live vector vaccine with chimeric avian influenza antigen epitopes as a supplementary vaccine to the existing avian influenza vaccine for the prevention and control of avian influenza. Summary of the Invention

[0006] The purpose of the present invention is to overcome the above-mentioned deficiencies in the prior art and to provide an attenuated duck Tembusu virus strain and its application.

[0007] The first object of the present invention is to provide an attenuated strain of duck Tembusu virus.

[0008] The second object of the present invention is to provide the use of the above-mentioned attenuated duck Tembusu virus strain in the preparation of a vaccine for immunizing duck Tembusu virus.

[0009] The third object of the present invention is to provide the use of the above-mentioned attenuated duck Tembusu virus strain in the preparation of live vector vaccine strains.

[0010] The fourth object of the present invention is to provide a method for preparing an attenuated strain of duck Tembusu virus.

[0011] The fifth object of the present invention is to provide a method for preparing a live vector vaccine strain.

[0012] The sixth object of the present invention is to provide a live vector vaccine strain prepared by the above preparation method.

[0013] The seventh object of the present invention is to provide the use of the above-mentioned live vector vaccine strain in the preparation of live vector vaccines.

[0014] In order to achieve the above object, the present invention is implemented through the following scheme:

[0015] An attenuated duck Tembusu virus strain, the nucleotide sequence of which is shown in SEQ ID NO: 1.

[0016] The present invention also seeks to protect the use of the attenuated duck Tembusu virus strain in preparing a vaccine for immunizing duck Tembusu virus.

[0017] The present invention also seeks to protect the use of the attenuated duck Tembusu virus strain in the preparation of live vector vaccine strains.

[0018] Preferably, the live vector vaccine strain is a live vector vaccine strain against avian influenza virus.

[0019] More preferably, the avian influenza virus is an H5N6 subtype avian influenza virus.

[0020] The present invention also claims a method for preparing an attenuated strain of duck Tembusu virus, comprising the following steps:

[0021] S1. Using the wild-type duck Tembusu virus S132 full-length cDNA infectious clone plasmid pB-S132-AH as a template, the EM-1 upstream primer (SEQ ID NO: 2) and the EM-1 downstream primer (SEQ ID NO: 3) were used to amplify the EM-1 fragment, and the EM-2 upstream primer (SEQ ID NO: 4) and the EM-2 downstream primer (SEQ ID NO: 5) were used to amplify the EM-2 fragment.

[0022] EM-1 and EM-2 fragments as templates, using the EM-1 upstream primer with the nucleotide sequence as shown in SEQ ID NO: 2 and the EM-2 downstream primer with the nucleotide sequence as shown in SEQ ID NO: 5 for amplification to obtain an EM fragment;

[0023] S2. Using the EM fragment obtained in step S1 as a template, using the EMT-1 upstream primer with the nucleotide sequence as shown in SEQ ID NO: 6 and the EMT-1 downstream primer with the nucleotide sequence as shown in SEQ ID NO: 7 for amplification to obtain an EMT-1 fragment, and using the EMT-2 upstream primer with the nucleotide sequence as shown in SEQ ID NO: 8 and the EMT-2 downstream primer with the nucleotide sequence as shown in SEQ ID NO: 9 for amplification to obtain an EMT-2 fragment;

[0024] Using the EMT-1 fragment and the EMT-2 fragment as templates, using the EMT-1 upstream primer with the nucleotide sequence as shown in SEQ ID NO: 6 and the EMT-2 downstream primer with the nucleotide sequence as shown in SEQ ID NO: 9 for amplification to obtain an EMT fragment;

[0025] S3. Using the EMT fragment obtained in step S2 as a template, using the ESMT-1 upstream primer with the nucleotide sequence as shown in SEQ ID NO: 10 and the ESMT-1 downstream primer with the nucleotide sequence as shown in SEQ ID NO: 11 for amplification to obtain an ESMT-1 fragment, and using the ESMT-2 upstream primer with the nucleotide sequence as shown in SEQ ID NO: 12 and the ESMT-2 downstream primer with the nucleotide sequence as shown in SEQ ID NO: 13 for amplification to obtain an ESMT-2 fragment;

[0026] Using the ESMT-1 fragment and the ESMT-2 fragment as templates, using the ESMT-1 upstream primer with the nucleotide sequence as shown in SEQ ID NO: 10 and the ESMT-2 downstream primer with the nucleotide sequence as shown in SEQ ID NO: 13 for amplification to obtain an ESMT fragment;

[0027] S4. Using Xba I endonuclease and Xho I endonuclease to digest the wild-type duck Tembusu virus S132 full-length cDNA infectious clone plasmid pB-S132-A-H to obtain linearized pB-S132-A-H;

[0028] Homologously recombining the ESMT fragment obtained in step S3 into the linearized pB-S132-A-H to obtain a recombinant plasmid pB-rS132-E SMT ;

[0029] S5. Using the recombinant plasmid pB-rS132-E obtained in step S4 as a template, using the EMT-1 upstream primer with the nucleotide sequence as shown in SEQ ID NO: 6 and the EMT-1 downstream primer with the nucleotide sequence as shown in SEQ ID NO: 7 for amplification to obtain an EMT-1 fragment, and using the EMT-2 upstream primer with the nucleotide sequence as shown in SEQ ID NO: 8 and the EMT-2 downstream primer with the nucleotide sequence as shown in SEQ ID NO: 9 for amplification to obtain an EMT-2 fragment;SMT The virus was transfected into cells and the virus solution was collected to obtain the attenuated duck Tembusu virus strain with a nucleotide sequence as shown in SEQ ID NO: 1.

[0030] The wild-type duck Tembusu virus S132 full-length cDNA infectious clone plasmid pB-S132-AH described in step S1 is disclosed in the prior art CN116837028A.

[0031] Preferably, the cells in step S5 are BHK-21 cells.

[0032] The present invention constructs an attenuated duck Tembusu virus strain based on reverse genetic technology, and uses it as a live vector to develop a live vector vaccine strain of chimeric virus antigens.

[0033] The present invention also claims a method for preparing a live vector vaccine strain, which is as follows:

[0034] The recombinant plasmid pB-rS132-E was cleaved by Pac I endonuclease and Xba I endonuclease. SMT Enzyme digestion was performed to obtain linearized pB-rS132-E SMT ;

[0035] The nucleotide sequence of PX-HAbNPt is shown in SEQ ID NO: 14 and homologously recombined into the linearized pB-rS132-E SMT The recombinant duck Tembusu virus full-length cDNA infectious clone plasmid pB-rS132-E was obtained. SMT -HAbNPt;

[0036] Using the recombinant duck Tembusu virus full-length cDNA infectious cloning plasmid pB-rS132-E SMT After HAbNPt transfects cells, the virus fluid is collected to obtain the live vector vaccine strain.

[0037] The present invention also seeks to protect the live vector vaccine strain prepared by the above preparation method.

[0038] Preferably, the nucleotide sequence of the live vector vaccine strain is shown in SEQ ID NO: 15.

[0039] The present invention also seeks to protect the use of the above live vector vaccine strain in the preparation of live vector vaccines.

[0040] Preferably, the live vector vaccine is a live vector vaccine against avian influenza virus.

[0041] More preferably, the avian influenza virus is an H5N6 subtype avian influenza virus.

[0042] Compared with the prior art, the application has the following beneficial effects:

[0043] The application provides a duck Tembusu virus attenuated strain, and the nucleotide sequence of the duck Tembusu virus attenuated strain is shown in SEQ ID NO: 1. The duck Tembusu virus attenuated strain can immunize ducklings and enable the ducklings to produce effective duck Tembusu virus antibodies, the protection rate of the ducklings reaches 100 %, meanwhile, the weight growth of the ducklings is not affected, and there is no virus discharge, death and viremia, and the duck Tembusu virus is not detected in the main organs of the ducklings. The duck Tembusu virus attenuated strain can also be used as a live vector to chimerize H5N6 subtype avian influenza virus antigen epitopes and further construct a live vector vaccine strain, and the live vector vaccine strain can immunize ducklings and effectively reduce the damage of H5N6 subtype avian influenza virus to the ducklings. BRIEF DESCRIPTION OF DRAWINGS

[0044] Figure 1 A weight analysis result graph of each group of SPF ducklings in Example 2;

[0045] Figure 2 A survival rate result graph of each group of ducklings in Example 2;

[0046] Figure 3 A clinical symptom result graph of each group of ducklings in Example 2; A is a feces observation graph of each group of ducklings on the 4th day after infection, B is a feed observation graph of each group of ducklings on the 3rd day after infection, and C is a brain dissection graph of each group of ducklings;

[0047] Figure 4 A detection result graph of virus replication levels in organs of each group of ducklings in Example 2;

[0048] Figure 5 A viremia detection result graph of serum of each group of SPF ducklings in Example 2;

[0049] Figure 6 A DTMUV antibody detection result graph of each group of ducklings in Example 2;

[0050] Figure 7 A weight test result graph of each group of SPF ducklings during an immunoprotection test in Example 2;

[0051] Figure 8 A clinical symptom result graph of each group of SPF ducklings during an immunoprotection test in Example 2; A is a feces observation graph of each group of ducklings on the 4th day after infection, B is a feed observation graph of each group of ducklings on the 4th day after infection;

[0052] Figure 9 A viremia detection result graph of each group of SPF ducklings during an immunoprotection test in Example 2;

[0053] Figure 10 Figure of test results of virus replication level in organs of each group of SPF ducklings during immune protection test in Example 2;

[0054] Figure 11 Figure of structural diagram of HAbNPt tandem epitope in Example 3;

[0055] Figure 12 Figure of test results of IFN-γ and IL-4 levels in Example 4; A is a figure of test results of IFN-γ level, and B is a figure of test results of IL-4 level;

[0056] Figure 13 Figure of test results of avian influenza virus neutralizing antibody titers of each group of SPF ducklings in Example 4;

[0057] Figure 14 Figure of analysis results of body weight of each group of SPF ducklings during H5N6 subtype avian influenza virus S230 challenge in Example 4;

[0058] Figure 15 Figure of survival rate of each group of SPF ducklings during H5N6 subtype avian influenza virus S230 challenge in Example 4;

[0059] Figure 16 Figure of test results of clinical symptoms of each group of SPF ducklings during H5N6 subtype avian influenza virus S230 challenge in Example 4; A is a figure of observation of eye cornea of SPF ducklings, and B is a figure of dissection observation of SPF ducklings;

[0060] Figure 17 Figure of test results of virus replication level in organs of each group of SPF ducklings during H5N6 subtype avian influenza virus S230 challenge in Example 4. DETAILED DESCRIPTION

[0061] The application will be further described below in conjunction with the drawings and specific examples in the specification, which are only used to explain the application and are not used to limit the scope of the application. The test methods used in the following examples are conventional methods unless otherwise specified, and the materials, reagents, etc. used are commercially available reagents and materials unless otherwise specified.

[0062] The wild-type duck Tembusu virus S132 full-length cDNA infectious clone plasmid pB-S132-A-H in the example of the application is disclosed in the prior art CN116837028A;

[0063] The wild-type duck Tembusu virus S132 full-length cDNA in the example of the application is disclosed in the prior art CN116837028A;

[0064] The feeds involved in the embodiments of the present invention are all duck feeds purchased from Zhengda Company;

[0065] The virus experimental operations involved in the examples of the present invention were performed in biosafety level 2 and 3 laboratories (BSL-2 and BSL-3 / ABSL-3 laboratories), and all animal experiments and animal experimental protocols were approved by the Experimental Animal Ethics Committee of South China Agricultural University.

[0066] Example 1 Recombinant plasmid pB-rS132-E for producing attenuated duck Tembusu virus strain SMT Construction

[0067] 1. Recombinant plasmid pB-rS132-E SMT Construction

[0068] S1. Using the wild-type duck Tembusu virus S132 full-length cDNA infectious clone plasmid pB-S132-AH as a template, the EM-1 upstream primer (SEQ ID NO: 2) and the EM-1 downstream primer (SEQ ID NO: 3) were used to amplify the EM-1 fragment, and the EM-2 upstream primer (SEQ ID NO: 4) and the EM-2 downstream primer (SEQ ID NO: 5) were used to amplify the EM-2 fragment.

[0069] Using the EM-1 fragment and the EM-2 fragment as templates, the EM-1 upstream primer having a nucleotide sequence as shown in SEQ ID NO: 2 and the EM-2 downstream primer having a nucleotide sequence as shown in SEQ ID NO: 5 were used to amplify the EM fragment;

[0070] S2. Using the EM fragment obtained in step S1 as a template, amplifying the EMT-1 fragment using an EMT-1 upstream primer with a nucleotide sequence as shown in SEQ ID NO: 6 and an EMT-1 downstream primer with a nucleotide sequence as shown in SEQ ID NO: 7 to obtain an EMT-1 fragment, and amplifying the EMT-2 fragment using an EMT-2 upstream primer with a nucleotide sequence as shown in SEQ ID NO: 8 and an EMT-2 downstream primer with a nucleotide sequence as shown in SEQ ID NO: 9;

[0071] Using the EMT-1 fragment and the EMT-2 fragment as templates, amplification was performed using an EMT-1 upstream primer having a nucleotide sequence as shown in SEQ ID NO: 6 and an EMT-2 downstream primer having a nucleotide sequence as shown in SEQ ID NO: 9 to obtain the EMT fragment;

[0072] S3. Using the EMT fragment obtained in step S2 as a template, amplifying the ESMT-1 fragment using an ESMT-1 upstream primer with a nucleotide sequence as set forth in SEQ ID NO: 10 and an ESMT-1 downstream primer with a nucleotide sequence as set forth in SEQ ID NO: 11, and amplifying the ESMT-2 fragment using an ESMT-2 upstream primer with a nucleotide sequence as set forth in SEQ ID NO: 12 and an ESMT-2 downstream primer with a nucleotide sequence as set forth in SEQ ID NO: 13;

[0073] Using the ESMT-1 fragment and the ESMT-2 fragment as templates, amplification was performed using an ESMT-1 upstream primer having a nucleotide sequence as shown in SEQ ID NO: 10 and an ESMT-2 downstream primer having a nucleotide sequence as shown in SEQ ID NO: 13 to obtain the ESMT fragment;

[0074] The specific amplification reaction system and amplification reaction procedure involved in steps S1 to S3 are shown in Table 1;

[0075] Table 1 Amplification reaction system and amplification reaction procedure

[0076]

[0077]

[0078] S4. The wild-type duck Tembusu virus S132 full-length cDNA infectious clone plasmid pB-S132-AH was digested with Xba I endonuclease and Xho I endonuclease to obtain linearized pB-S132-AH (wherein the linearized pB-S132-AH is linearized and the gene sequence between the Xba I restriction site and the Xho I restriction site is removed).

[0079] The ESMT fragment obtained in step S3 was homologously recombined into the linearized pB-S132-AH to obtain the recombinant plasmid pB-rS132-E SMT .

[0080] 2. Attenuated duck Tembusu virus strain rS132-E SMT Construction

[0081] The pB-rS132-E prepared in step 1 of Example 1 was SMT The attenuated strain rS132-E of duck Tembusu virus was obtained by plaque purification after transfection into BHK-21 cells and cultured for 3 days. SMT .

[0082] Example 2 attenuated strain of duck Tembusu virus rS132-E SMT pathogenicity and immune protection test

[0083] I. Experimental method

[0084] 1. Pathogenicity test

[0085] (1) Experimental group setting

[0086] Thirty-three SPF ducklings were randomly divided into three groups, namely the DMEM group (DMEM cell culture fluid group as the negative control group), the S132 group and the rS132-E SMT group, 11 in each group (3 for anatomical analysis and 8 for evaluation analysis).

[0087] S132 group: 11 SPF ducklings were infected with wild-type duck Tembusu virus S132 at an infection dose of 10 4 PFU / 0.2 mL per duckling.

[0088] rS132-E SMT group: 11 SPF ducklings were infected with rS132-E 4 obtained in Example 1 with the nucleotide sequence shown in SEQ ID NO: 1 at an infection dose of 10 SMT PFU / 0.2 mL per duckling.

[0089] DMEM negative control group: The only difference from the S132 group is that the wild-type duck Tembusu virus S132 is replaced with an equal amount of DMEM cell culture fluid to inoculate SPF ducklings.

[0090] The S132 group, the rS132-E SMT group and the DMEM negative control group all inoculate SPF ducklings by intramuscular injection, and the pathogenicity test period is 14 days.

[0091] (2) Excretion test

[0092] During the pathogenicity test, cloacal swabs of 8 ducklings for evaluation analysis were collected from each group of SPF ducklings every 2 days after infection (2nd, 4th, 6th, 8th, 10th, 12th and 14th day after infection), and cDNA was obtained by reverse transcription using PrimeScript TM II 1st Strand cDNA Synthesis Kit kit (TAKARA, item number 6210A) according to the kit instructions.

[0093] Using the cDNA obtained by reverse transcription as a template, fluorescent quantitative PCR was performed using NS5-F with a nucleotide sequence as shown in SEQ ID NO: 16 and NS5-R with a nucleotide sequence as shown in SEQ ID NO: 17 according to the DTMUV fluorescent quantitative PCR amplification system shown in Table 2 (each sample was repeated three times, i.e., three CT values ​​were obtained for each sample), and the average CT value of each sample was calculated.

[0094] Table 2DTMUV fluorescence quantitative PCR amplification system

[0095]

[0096] Amplification program: 95°C, 2 min; 95°C, 15 s, 58°C, 30 s, 40 cycles.

[0097] Judgment criteria: 0<average CT value ≤ 35, the sample is judged as DTMUV positive; average CT value > 35, the sample is judged as DTMUV negative.

[0098] Standard curve establishment and calculation of DTMUV RNA copy number in positive products: The standard plasmid (pMD19T plasmid containing DTMUV NS5 gene) was diluted with ddH2O to obtain a copy number of 10 6 copies / μL, 10 5 copies / μL, 10 4 copies / μL, 10 3 copies / μL, 10 2 The standard plasmids at various dilutions with concentrations of 10 copies / μL and 10 copies / μL were used as templates. Fluorescence quantitative PCR was performed according to the DTMUV fluorescence quantitative PCR amplification system shown in Table 2 and the CT value was tested. Each dilution was repeated 3 times (that is, 3 corresponding CT values ​​were obtained by DTMUV fluorescence quantitative PCR for each dilution of the standard plasmid), and the average CT value was taken.

[0099] The copy number index of each dilution of the standard plasmid is used as the horizontal axis (e.g. the copy number is 10 6 copies / μL, the corresponding value on the horizontal axis is 6, i.e. x=6), and the average CT value (y value) of the standard plasmids with different dilutions is the vertical axis. A standard curve is established as shown in Formula I:

[0100] Formula I: y = -3.5489x + 40.788.

[0101] The average CT value of the DTMUV-positive samples was used as the y value and substituted into the standard curve shown in Formula I to obtain the corresponding x value. The DTMUV RNA copy number in the DTMUV-positive samples was 2×10 xcopies / μL.

[0102] (3) Duckling weight analysis

[0103] During the pathogenicity test, feed was added to the feed trough at 9:00 a.m. every day at a rate of 100 g per duck, and the residual feed in the feed trough was removed when feeding the ducks every other day. Water was fed regularly at 9:00 a.m. every day and 6:00 p.m. every day. The weights of the eight ducklings in each group of SPF ducklings used for evaluation and analysis were recorded, and the average value was taken as the weight of the ducklings in that group.

[0104] (4) Duckling survival rate and clinical symptom detection

[0105] During the pathogenicity test, the clinical symptoms and mortality of 8 ducklings in each group of SPF ducklings used for evaluation and analysis were recorded every day; the clinical symptoms included: defecation, diet and water intake, mental state and posture.

[0106] (5) Virus replication level test in duckling organs

[0107] During the pathogenicity test, on the third day after infection in each group, three SPF ducklings from each group were dissected for anatomical analysis, and the hearts, livers, spleens, lungs, kidneys, and brains of the SPF ducklings from each group were collected for plaque titration, specifically:

[0108] The collected SPF duckling hearts were minced and placed in a 2 mL EP tube. PBS containing 10,000 units of penicillin and streptomycin was added to the EP tube at a ratio of 1 g:1 mL, and autoclaved steel balls were placed in the tube. The tube was ground for 10 min using a low-temperature high-throughput tissue grinder, and then placed in a -80 °C refrigerator and frozen and thawed three times. The tube was then centrifuged at 1000 rpm / min and 4 °C for 10 min, and the supernatant was collected.

[0109] The collected supernatants were divided into 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 and 10 -6 Dilute the virus solution at different dilution multiples to obtain virus solutions of different dilutions.

[0110] BHK-21 cells were plated in cell plates (6-well) and cultured with DMEM cell culture medium. When the BHK-21 cells in each well grew to 90% of the well area, the cell culture medium in the cell plates was discarded, and the cells were washed twice with PBS. Then, each dilution of the virus solution was inoculated into the cell plates at 0.4 mL / well (3 wells for each dilution of the virus solution), and the cell plates inoculated with the virus solution were incubated in a cell culture incubator at 37°C, 5% (v / v) CO2for 1 h, with shaking every 20 min. After incubation, the supernatant was discarded, and an agar overlay layer {containing 1 mL of 1.8% (w / w) agarose, 0.04 mL of FBS, 0.02 mL of 100x penicillin-streptomycin double antibody (Gibco, 15140-122), and 0.94 mL of 2x DMEM} was added to the cell plates at 2 mL / well. The cell plates were placed at 25°C for 25 min, and then inverted and placed in a cell culture incubator at 37°C, 5% (v / v) CO2for culture until visible plaques appeared in the cell plates. The plaques were fixed with 4% (v / v) paraformaldehyde for 12 h, and then stained with crystal violet for 20 min after removing the agar overlay layer. The number of plaques in the cell wells inoculated with each dilution of the virus solution was recorded. The cell wells with virus solution at a dilution with ≤20 plaques, which were clear and easy to count, were used to calculate the PFU according to Formula II, and the virus replication level in the heart of the SPF duckling 3 days after infection was obtained.

[0111] Formula II: PFU = a * b / v, wherein PFU represents the plaque forming units per milliliter of the virus sample, a represents the plaque mean (i.e., the average number of plaques appearing in the repeated well positions of the virus solution at the same dilution), b is the reciprocal of the dilution, and v = the volume (mL) of the virus solution inoculated at each dilution.

[0112] The liver, spleen, lung, kidney, and brain of the collected SPF duckling were treated in the same manner, and the virus replication levels in the liver, spleen, lung, kidney, and brain of the SPF duckling 3 days after infection were calculated.

[0113] (6) Detection of DTMUV antibodies in ducklings

[0114] During the pathogenicity test, the serum of 8 ducklings for evaluation and analysis was collected from each group of SPF ducklings on day 1, day 3, day 7, and day 14 after infection, and the DTMUV copy number and antibody level in the serum were detected, as follows:

[0115] The DTMUV RNA copy number in the serum of each group of SPF ducklings on day 1 and day 3 after infection was detected according to the method shown in step (2), and whether viremia occurred in each group of SPF ducklings was determined.

[0116] A DTMUV antibody ELISA quantitative kit (Jiangsu Enzyme Immunity Industry Co., Ltd., catalog number MM-92575901) was used to detect the DTMUV antibody content in the serum of each group of SPF ducklings according to the kit instructions.

[0117] 2. Immune protection test

[0118] (1) Experimental treatment

[0119] Use the rS132-E in the pathogenicity test experiment shown in step 1 SMT The immune protection test was performed on 8 SPF ducklings in the group and the DMEM negative control group for evaluation analysis. In the pathogenicity experiment, the 14th day after virus infection was recorded as the 0th day of infection. SMT The SPF ducklings in the group and the DMEM negative control group were treated with wild strain S132 at a rate of 10 5 The dose of PFU / 0.2mL was injected intramuscularly for challenge treatment. The challenge test period was 14 days. Among the 8 SPF ducklings in each group, 3 were used to analyze the replication level of the wild strain S132 in the organs of the ducklings after challenge, and 5 were used for evaluation and analysis.

[0120] (2) Detoxification test

[0121] During the immune protection test, cloacal swabs of 8 ducklings in each group for evaluation and analysis were collected every 2 days (2, 4, 6, 8, 10, 12 and 14 days after the challenge) and the PrimeScript TM II 1st Strand cDNA Synthesis Kit (TAKARA, Cat. No. 6210A) was used for reverse transcription according to the kit instructions to obtain cDNA of the immune protection test ducklings.

[0122] Using the cDNA obtained by reverse transcription as a template (sample), fluorescent quantitative PCR amplification was performed using the nucleotide sequence of NS5-F shown in SEQ ID NO: 16 and the nucleotide sequence of NS5-R shown in SEQ ID NO: 17 according to the DTMUV fluorescent quantitative PCR amplification system shown in Table 2 (each sample was repeated three times, i.e., three CT values ​​were obtained for each sample), and the average CT value of each sample was calculated.

[0123] Judgment criteria: 0<average CT value ≤ 35, the sample is judged as DTMUV positive; average CT value > 35, the sample is judged as DTMUV negative.

[0124] Substitute the average CT value (i.e., y value) of the positive product into the standard curve shown in Formula I to obtain the corresponding x value. The DTMUV RNA copy number in the positive product is 2×10 xcopies / μL.

[0125] (3) Duckling weight test

[0126] During the immune protection test, feed was added to the feed trough at 9 a.m. every day at a rate of 100 g per duck, and the residual feed in the feed trough was removed when feeding the ducks every other day. Water was fed regularly at 9 a.m. every day and 6 p.m. every day. The weights of the five ducklings in each group of SPF ducklings used for evaluation and analysis were recorded, and the average value was taken as the weight of the ducklings in that group.

[0127] (4) Clinical symptom detection of ducklings

[0128] During the immune protection test, the clinical symptoms of 5 ducklings in each group for evaluation and analysis were recorded every day; the clinical symptoms included: defecation, diet and water intake, mental state and posture.

[0129] (5) Virus replication level test in duckling organs

[0130] According to the test method for virus replication level in duckling organs shown in step 1, the virus replication level of the heart, liver, spleen, lung, kidney and brain of each group of SPF ducklings was measured on the third day after the infection.

[0131] 2. Experimental Results

[0132] 1. Pathogenicity test results

[0133] (1) Detoxification test results

[0134] The test results of DTMUV RNA copy number of ducklings in each group are shown in Table 3.

[0135] Table 3 DTMUV RNA copy number test results of ducklings in each group

[0136]

[0137]

[0138] Note: a represents the total number of ducklings / ducklings that excrete toxins; b represents the average number of DTMUV RNA copies (Log 10 copies / μL)±standard deviation.

[0139] The results showed that one SPF duckling in the S132 group excreted toxins (i.e., DTMUV RNA copies were detected) on the second day after infection with the wild strain S132, four SPF ducklings excreted toxins on the fourth day after infection, and eight SPF ducklings excreted toxins on the sixth day after infection. The excretion rate of the SPF ducklings in the S132 group was 100%, with the highest excretion rate on the fourth day after infection, which was 101.92 copies / μL.

[0140] rS132-E SMT In the SPF ducklings of the group, only one SPF duckling was found to have excretion of toxins on the second day after infection, with an excretion rate of 12.5% ​​and an excretion amount of 10 1.81 copies / μL, and the detoxification rate was reduced by 87.5% compared with the S132 group.

[0141] (2) Duckling weight analysis results

[0142] The weight analysis results of each group of SPF ducklings are shown in the figure below: Figure 1 As shown, the results showed that: rS132-E SMT The weight of the SPF ducklings in the rS132-E group and the DMEM negative control group increased steadily over time, while the weight of the SPF ducklings in the S132 group increased slowly from 3 to 10 days after infection with the wild-type strain S132. The average weight of the SPF ducklings in the DMEM group on the 14th day after inoculation with DMEM cell culture medium increased by about 2.4 times compared with the average weight on the 0th day. SMT The SPF ducklings in the group were infected with rS132-E shown in SEQ ID NO: 1. SMT The average body weight of SPF ducklings in the S132 group on the 14th day after infection with the wild strain S132 increased by about 2 times compared with the average body weight on the 0th day.

[0143] Results: The nucleotide sequence is shown in SEQ ID NO: 1 as rS132-E SMT There was no effect on the growth of SFP ducklings.

[0144] (3) Duckling survival rate and clinical symptom detection results

[0145] The survival rate of ducklings in each group is shown in the figure below. Figure 2 The results showed that one SPF duckling died during the pathogenicity test (died on the 7th day after infection with the wild strain S132) in the S132 group, and seven survived, with a survival rate of 87.5% for the SPF ducklings. SMT All SPF ducklings in the group survived during the pathogenicity experiment, with a survival rate of 100%.

[0146] Results: In each 10 4 At an infection dose of PFU / 0.2 mL, the nucleotide sequence obtained in Example 1 is shown in SEQ ID NO: 1 as rS132-E SMT No ducklings were killed.

[0147] The clinical symptom test results of each group of ducklings are shown in the figure below: Figure 3 As shown, A is the feces observation picture of each group of ducklings on the 4th day after infection, B is the feed observation picture of each group of ducklings on the 3rd day after infection, and C is the brain autopsy picture of each group of ducklings.

[0148] The results showed that the SPF ducklings in the S132 group began to excrete green loose feces on the 4th day after being infected with the wild-type S132 strain, while the DMEM negative control group and rS132-E SMT The SPF ducklings in the S132 group showed normal performance (the feces were brown-gray with white urate near one end); and the SPF ducklings in the S132 group showed symptoms of decreased appetite on the third day after infection with the wild-type S132, while the DMEM negative control group and rS132-E SMT The ducklings in the two groups had normal appetite after infection.

[0149] The brain autopsy results of the SPF ducklings in each group showed that the brains of the SPF ducklings that died on the 7th day after infection in the S132 group showed more obvious hemorrhage symptoms than those of the SPF ducklings that died on the 3rd day after infection in the S132 group; SMT No abnormalities were found in the brains of the SPF ducklings in the group.

[0150] Results: The nucleotide sequence obtained in Example 1 is shown in SEQ ID NO: 1 as rS132-E SMT After infection of SPF ducklings, no obvious clinical symptoms appeared.

[0151] (4) Detection results of viral replication levels in the organs of ducklings in each group

[0152] The results of the detection of virus replication levels in the organs of ducklings in each group are shown in the figure below. Figure 4 The results showed that on the third day after infection, DTMUV was detected in the heart, liver, spleen, lung, kidney and brain of SPF ducklings in the S132 group, with an average titer of 10 3.79 PFU / mL, 10 3.77 PFU / mL, 10 3.74 PFU / mL, 10 4.48 PFU / mL, 10 3.56 PFU / mL and 10 2.35 PFU / mL; rS132-E SMT DTMUV was detected in the liver, spleen, lung and kidney of SPF ducklings in the group with an average titer of 10 0.99 PFU / mL, 10 2.94 PFU / mL, 10 1.47 PFU / mL and 10 0.74PFU / mL, while DTMUV was not detected in the heart and brain, and the average titers of each organ where DMTUV was detected were significantly lower than those in the SPF ducklings in the S132 group.

[0153] Results: The nucleotide sequence obtained in Example 1 is shown in SEQ ID NO: 1 as rS132-E SMT After infection of SPF ducklings, the virus titers in the main organs (heart, liver, spleen, lung, kidney and brain) of SPF ducklings were significantly lower than those in the main organs of SPF ducklings infected with wild strain S132, indicating that the nucleotide sequence obtained in Example 1 is as shown in SEQ ID NO: 1 rS132-E SMT It is an attenuated strain.

[0154] (5) Viremia test results and DTMUV antibody test results of ducklings in each group

[0155] The results of viremia test of SPF duckling serum in each group are shown in the figure below: Figure 5 As shown, the results showed that no DTMUV RNA was detected in the serum of SPF ducklings in the DMEM negative control group on the 1st and 3rd day after inoculation with DMEM cell culture medium.

[0156] The serum of 8 SPF ducklings in the S132 group was detected with DTMUV RNA on the first day after infection with the wild strain S132, with an average copy number of 10 4.5 copies / μL; on the third day after infection, DTMUV RNA was detected in the serum of 5 SPF ducklings in the S132 group, with an average copy number of 10 3.1 copies / μL.

[0157] rS132-E SMT In the group infected with rS132-E SMT On the first day after incubation, only one SPF duckling was detected in the serum with a copy number of 10 0.99 copies / μL, which was much lower than the average DTMUV RNA copy number in the S132 group on the first day after SPF ducklings were infected with wild strain S132; on the third day after infection, rS132-E SMT No DTMUV RNA was detected in the serum of SPF ducklings in group A.

[0158] Results: The nucleotide sequence obtained in Example 1 is shown in SEQ ID NO: 1 as rS132-E SMT After infection of SPF ducklings, only one SPF duckling showed mild viremia on the first day after infection, and no SPF ducklings showed viremia as the infection time increased.

[0159] The DTMUV antibody detection results of the ducklings in each group are shown in the following table. Figure 6 As shown in the table, the results show that the average levels of DTMUV antibodies in the serum of SPF ducklings in the DMEM negative control group after inoculation with DMEM cell culture solution on the 1st day, the 3rd day, the 7th day and the 14th day were 51.18 pg / mL, 55.19 pg / mL, 59.7 pg / mL and 53.46 pg / mL respectively; the average levels of DTMUV antibodies in the serum of SPF ducklings in the S132 group after inoculation with wild strain S132 on the 1st day, the 3rd day, the 7th day and the 14th day were 53.4 pg / mL, 73.7 pg / mL, 101.85 pg / mL and 103.6 pg / mL respectively; the average levels of DTMUV antibodies in the serum of SPF ducklings in the rS132-E SMT group after inoculation with the rS132-E SMT group after inoculation with the rS132-E

[0160] In summary, compared with infection with wild strain S132, the rS132-E SMT After infection of SPF ducklings, the body weight of the ducklings stably increased, the clinical symptoms were not obvious, the survival rate was 100%, the virus shedding rate was significantly reduced (all reduced by 87.5%), the virus replication level in the main organs was significantly reduced (P<0.001), and viremia was basically not detected after the 1st day and the 3rd day of infection (only the rS132-E SMT group 1 duckling after infection with virus on the 1st day, P<0.001), and the rS132-E SMT After the 14th day of infection with virus, the ducklings could produce a higher level of DTMUV antibodies.

[0161] Therefore, the rS132-E SMT can be used as a live attenuated vaccine candidate strain to immunize ducklings.

[0162] 2. Immune protection test results

[0163] (1) Virus shedding test results

[0164] The DTMUV RNA copy number test results of the ducklings in each group after challenge are shown in Table 4.

[0165] Table 4 DTMUV RNA copy number test results of ducklings in each group after challenge

[0166]

[0167] Note: a represents the total number of ducklings / ducklings that excrete toxins; b represents the average number of DTMUV RNA copies (Log 10 copies / μL)±standard deviation.

[0168] The results showed that among the 5 SPF ducklings in the DMEM negative control group, 3 ducklings were detected to excrete toxins on the 2nd day after the challenge, 5 ducklings were detected to excrete toxins on the 4th day after the challenge, 5 ducklings were detected to excrete toxins on the 6th day after the challenge, and 1 duckling was detected to excrete toxins on the 8th day after the challenge. The average excretion of toxins on the 4th day after the challenge was the highest, which was 10 2.72 copies / μL; and rS132-E SMT No toxin excretion was detected in the 5 SPF ducklings in the group.

[0169] (2) Duckling weight test results

[0170] The weight test results of each group of SPF ducklings during the immune protection test are shown in the figure below: Figure 7 The results showed that the weight gain of SPF ducklings in the DMEM group slowed down from 4 to 9 days after the challenge, and decreased significantly from 4 to 5 days. The average weight on the 14th day after the challenge increased by 1.37 times compared with the average weight on the 0th day after the challenge. SMT The weight of SPF ducklings in the group increased steadily after the infection, and the average weight on the 14th day after the infection increased by 1.48 times compared with the average weight on the 0th day after the infection.

[0171] Results: The nucleotide sequence obtained in Example 1 is shown in SEQ ID NO: 1 as rS132-E SMT The average weight growth rate of immunized SPF ducklings after challenge with wild strain S132 was higher than that of SPF ducklings inoculated with DMEM cell culture medium.

[0172] (3) Clinical symptom test results of ducklings

[0173] The clinical symptom test results of SPF ducklings in each group during the immune protection test are shown in the figure below: Figure 8 As shown, A is the feces observation picture of each group of ducklings on the 4th day after the poison challenge, and B is the feed observation picture of each group of ducklings on the 4th day after the poison challenge.

[0174] The results showed that the SPF ducklings in the DMEM negative control group had green loose feces on the 4th day after the infection, while the rS132-E SMTThe SPF ducklings in the DMEM negative control group showed normal performance (feces were brown-gray with white urate near one end); and the SPF ducklings in the DMEM negative control group had a decreased appetite on the 4th day after the challenge, while the rS132-E SMT The SPF ducklings in the group had normal appetite.

[0175] (4) Viremia test results of ducklings

[0176] The results of viremia test of SPF ducklings in each group during the immune protection test are shown in the figure below: Figure 9 The results showed that on the first day after the challenge, DTMUV RNA was detected in the serum of the five ducklings in the DMEM negative control group, and the average copy number of DTMUV RNA was 10 4.21 On the third day after challenge, DTMUV RNA was detected in the serum of three ducklings in the DMEM group, with an average copy number of 10 2.53 copies / μL; and rS132-E SMT No DTMUV RNA was detected in the serum of group 5 ducklings on the 1st and 3rd day after infection.

[0177] Results: The nucleotide sequence obtained in Example 1 is shown in SEQ ID NO: 1 as rS132-E SMT The immunized SPF ducklings did not show viremia after being challenged with the wild strain S132.

[0178] (5) Virus replication level test results in duckling organs

[0179] The test results of virus replication levels in organs of SPF ducklings in each group during the immune protection test are shown in the figure below. Figure 10 The results showed that DTMUV was detected in the heart, liver, spleen, lung, kidney and brain of SPF ducklings in the DMEM negative control group on the 3rd day after the challenge, with an average titer of 10 3.12 PFU / mL, 10 2.98 PFU / mL, 10 3.83 PFU / mL, 10 3.72 PFU / mL, 10 2.75 PFU / mL and 10 1.74 PFU / mL; while rS132-E SMT No DTMUV was detected in the organs of SPF ducklings in the control group.

[0180] Based on the above results, compared with the SPF ducklings inoculated with DMEM cell culture medium (DMEM negative control group), the rS132-E SMTImmunized SPF ducklings (rS132-E SMT After being challenged with the wild-type S132 virus, the mice in the 4th group showed stable weight gain, no obvious clinical symptoms, no detoxification, no viremia, and no replication of the wild-type S132 virus was detected in their major organs.

[0181] Therefore, when ducklings are challenged with wild-type duck Tembusu virus, the nucleotide sequence obtained in Example 1 is shown in SEQ ID NO: 1 as rS132-E SMT It can provide 100% protection for ducklings and can be used as a genetically engineered attenuated live vaccine strain to immunize ducklings.

[0182] Example 3 Preparation of a H5N6 subtype avian influenza live vector vaccine strain

[0183] 1. Recombinant duck Tembusu virus full-length cDNA infectious clone plasmid pB-rS132-E SMT Preparation of -HAbNPt

[0184] The pB-rS132-E obtained in Example 1 was cleaved using Pac I endonuclease and Xba I endonuclease. SMT Enzyme digestion was performed to obtain linearized pB-rS132-E SMT (The linearized pB-rS132-E SMT The gene sequence between the Pac I restriction site and the Xba I restriction site was removed); the PX-HAbNPt sequence shown in SEQ ID NO: 14 was homologously recombined into the linearized pB-rS132-E SMT The recombinant duck Tembusu virus full-length cDNA infectious clone plasmid pB-rS132-E was obtained. SMT -HAbNPt.

[0185] The structure of PX-HAbNPt as shown in the nucleotide sequence of SEQ ID NO: 14 is PacI-CMV-5'UTR-C37-HAbNPt-2A-C-prM40-XbaI, where PacI and XbaI are restriction enzyme cleavage sites, CMV is the nucleotide sequence corresponding to the CMV promoter, 5'UTR is the nucleotide sequence of the 5' non-coding region of DTMUV, C37 is the nucleotide sequence corresponding to the first 37 amino acids of the C protein of DTMUV, HAbNPt is the nucleotide sequence corresponding to the tandem epitopes of the HA protein and NP protein of the H5N6 subtype avian influenza virus, 2A is the nucleotide sequence corresponding to the self-cleaving polypeptide T2A (derived from the iridium virus), C is the nucleotide sequence corresponding to the C protein of DTMUV, and prM 40 The nucleotide sequence corresponding to the first 40 amino acids of the prM protein of DTMUV.

[0186] The amino acid sequence of the tandem epitope HAbNPt of the HA protein and the NP protein of the H5N6 subtype avian influenza virus is shown in SEQ ID NO: 18, and the structural schematic diagram is shown in Figure 11 The tandem epitope HAbNPt of the HA protein and the NP protein of the H5N6 subtype avian influenza virus is expressed in the order of HA-B-1, HA-B-2, HA-B-3, NP-T-1 and NP-T-2, wherein the three B cell epitopes of the HA protein of the H5N6 subtype avian influenza virus are expressed in tandem through the flexible linker (GGGGS) 3 between HA-B-1, HA-B-2 and HA-B-3; the B cell epitope HA-B-3 of the HA protein of the H5N6 subtype avian influenza virus and the T cell epitope NP-T-1 of the NP protein of the H5N6 subtype avian influenza virus are expressed in tandem through the flexible linker (GGGGS) 3 and the target sequence KK of the lysosomal protease; the two T cell epitopes of the NP protein of the H5N6 subtype avian influenza virus are expressed in tandem through the protease preferential cleavage site AAY as a spacer sequence between NP-T-1 and NP-T-2; the information of HA-B-1, HA-B-2, HA-B-3, NP-T-1 and NP-T-2 is shown in Table 5.

[0187] Table 5 Information of HA-B-1, HA-B-2, HA-B-3, NP-T-1 and NP-T-2

[0188] Cellular linear epitopes Amino acid sequence Amino acid length HA-B-1 (SEQ ID NO: 19) RESTQKAIDGVTNKVNS 17 HA-B-2 (SEQ ID NO: 20) ERANPANDLCYPGNFNDYEELKH 23 HA-B-3 (SEQ ID NO: 21) DYPQYSEEARLKREEISGVKLETIGTFQI 29 NP-T-1 (SEQ ID NO: 22) RLIQNSITI 9 NP-T-2 (SEQ ID NO: 23) MVMELIRMI 9

[0189] II. H5N6 subtype avian influenza live vector vaccine strain rS132-E SMT Preparation of HAbNPt

[0190] BHK-21 cells were plated in a 6-well plate and cultured with DMEM cell culture medium. When the BHK-21 cells in each well grew to 80% of the well area, the cell culture medium in the 6-well plate was discarded, each well was washed twice with anti-free opti-MEM, and then anti-free opti-MEM was added at 800 μL / well to obtain the ready-to-use well plate.

[0191] Two 1.5 mL EP tubes were labeled as A tube and B tube, 100 μL of anti-free opti-MEM was added to each of the A tube and the B tube, then 2 μg of the recombinant duck Tembusu virus full-length cDNA infectious clone plasmid pB-rS132-E SMT Preparation of HAbNPt, 4 μL of Lipofecamine 2000 liposome was added to the B tube, each tube was mixed uniformly and then placed at 25°C for 5 min, then the liquid in the B tube was transferred to the A tube, mixed uniformly and then placed at 25°C for 20 min to obtain the incubation liquid.

[0192] The incubation solution was added to the standby well plate, and the plate was placed in a constant temperature incubator at 37°C and 5% (v / v) CO2 for incubation. The plate was shaken every 30 minutes. After incubation for 5 hours, the liquid in the standby well plate was discarded, and DMEM containing 2% FBS was added at a rate of 2 mL / well. The plate was placed in a constant temperature incubator at 37°C and 5% (v / v) CO2 for incubation for 3 days. The supernatant was collected for plaque purification to obtain the H5N6 subtype avian influenza live vector vaccine strain rS132-E as shown in SEQ ID NO: 15. SMT -HAbNPt.

[0193] Example 4 H5N6 subtype avian influenza live vector vaccine strain rS132-E SMT - Immune protection test of HAbNPt

[0194] 1. Experimental Methods

[0195] 1. Experimental group settings

[0196] Sixty healthy 2-week-old SPF ducklings were randomly divided into three groups, including a negative control group, an attenuated strain group, and a live vector vaccine strain group, with 20 ducks in each group (3 of which were used for anatomical analysis and 17 for evaluation analysis).

[0197] Attenuated strain group: 10 per 4 The dose of PFU / 0.2 mL was obtained using the rS132-E nucleotide sequence shown in SEQ ID NO: 1 obtained in Example 1. SMT SPF ducklings were immunized, and a second immunization (second immunization) was performed with the same dose 2 weeks after immunization.

[0198] Live vector vaccine strain group: 10 per 4 The dose of PFU / 0.2 mL was obtained using the H5N6 subtype avian influenza live vector vaccine strain rS132-E, whose nucleotide sequence is shown in SEQ ID NO: 15 as obtained in Example 3. SMT -HAbNPt was used to immunize SPF ducklings, and a second immunization was performed at the same dose 2 weeks after immunization (second immunization).

[0199] The only difference between the negative control group and the attenuated strain is that the nucleotide sequence obtained in Example 1, rS132-E shown in SEQ ID NO: 1, SMT The cell culture medium was replaced with an equal volume of DMEM, and the rest of the treatments were exactly the same.

[0200] Two weeks after the second vaccination, the SPF ducklings in the attenuated strain group, live vector vaccine strain group and negative control group were challenged with H5N6 subtype avian influenza virus S230 strain (NCBI GenBank accession number: PQ057042.1~PQ057049.1) at a challenge dose of 10 3 EID 50 / 0.2mL, the method of attacking the poison is eye drops and nasal drops.

[0201] 2. Antibody level test

[0202] Serum (serum samples) were collected once a week after immunization from the 17 SPF ducklings in the negative control group, attenuated strain group, and live vector vaccine strain group used for evaluation and analysis in step 1. The IFN-γ and IL-4 levels in the serum were detected using a duck IFN-γ quantitative detection kit (Jiangsu Enzyme Immunity Industrial Co., Ltd., catalog number MM-007301) and a duck IL-4 quantitative detection kit (Jiangsu Enzyme Immunity Industrial Co., Ltd., catalog number MM-9162001) according to the kit instructions, and the avian influenza virus neutralizing antibody titer in the serum samples was determined by neutralization test;

[0203] The neutralization test process is as follows:

[0204] (1) Add 50 μL of PBS to each well of a 96-well plate; inactivate the serum sample at 56°C for 30 min, and then perform serial 2-fold dilutions (2 -1 ~2 -6 ):The dilution is 2 -1 The serum samples were added into the A1 to F1 wells in the first column of the 96-well plate at 50 μL / well (6 replicates). The PBS in the A1 to F1 wells and the extinguished serum samples were mixed evenly with a dispenser. Then 50 μL was aspirated and added into the A2 to F2 wells in the second column, and so on. 50 μL was discarded from the A6 to F6 wells.

[0205] (2) After the serum was diluted, 50 μL of virus solution (H5N6 subtype avian influenza virus S230 strain, 100 TCID) was added to each well. 50 / 50 μL) and placed in a cell culture incubator at 37°C, 5% (v / v) CO2 for 1 h.

[0206] (3) When the serum and virus neutralization process was almost finished, the 96-well cell plate containing DF-1 cells prepared in advance was washed twice with PBS, and then the neutralized serum and virus mixture was transferred to the cell plate in sequence and placed in a cell culture incubator at 37°C and 5% (v / v) CO2 for incubation for 1 hour.

[0207] (4) After the incubation, the incubation solution in the cell plate was discarded, and DMEM containing 2% (w / w) FBS was added, and the cell plate was incubated at 37°C in a 5% (v / v) CO2 cell incubator for 72 h.

[0208] (5) After 72 h of culture, the supernatant of each well was collected for hemagglutination test (HA) to determine whether the cells in each well were infected with avian influenza virus, and the wells with cells not infected with avian influenza virus were marked, and the antibody neutralization titer was calculated by the Reed-Muench method; the experimental operation of the AI live virus was performed in the biosafety level 2 and 3 laboratories according to the regulations.

[0209] 3. Body weight analysis of ducklings

[0210] During the challenge with the H5N6 subtype avian influenza virus S230 strain, the ducklings were fed regularly and quantitatively every day, and the body weights of 17 ducklings for evaluation and analysis in each group of ducklings were recorded, and the average value was taken as the body weight of the ducklings in the group.

[0211] 4. Survival rate and clinical symptom detection of ducklings

[0212] During the challenge with the H5N6 subtype avian influenza virus S230 strain, the clinical symptoms and death of 17 SPF ducklings for evaluation and analysis in each group of SPF ducklings were recorded every day; the clinical symptoms included defecation, diet and water intake, mental state, and posture.

[0213] 5. Virus shedding test

[0214] During the challenge with the H5N6 subtype avian influenza virus S230 strain, cloacal swabs and oral swabs of 17 SPF ducklings for evaluation and analysis in each group were collected every 2 days after challenge (on days 2, 4, 6, 8, 10, 12, and 14 after challenge), and PrimeScript TM II 1st Strand cDNA Synthesis Kit kit (TAKARA, item number 6210A) was used for reverse transcription according to the kit instructions to obtain cDNA of cloacal swabs and cDNA of oral swabs.

[0215] The cDNA of cloacal swabs and the cDNA of oral swabs were used as templates (samples), H5N6-F with the nucleotide sequence as shown in SEQ ID NO: 24 and H5N6-R with the nucleotide sequence as shown in SEQ ID NO: 25 were used for fluorescence quantitative PCR amplification according to the H5N6 subtype avian influenza virus fluorescence quantitative PCR amplification system shown in Table 6 (each sample was repeated 3 times, i.e., 3 CT values were obtained for each sample), and the average CT value of each sample was calculated.

[0216] Judgment criteria: 0<average CT value ≤ 30, the sample is judged to be positive for H5N6 subtype avian influenza virus; average CT value > 30, the sample is judged to be negative for H5N6 subtype avian influenza virus.

[0217] Table 6 Fluorescence quantitative PCR amplification of H5N6 subtype avian influenza

[0218]

[0219] Amplification program: 95°C, 2 min; 95°C, 15 s, 56°C, 30 s, 40 cycles.

[0220] 6. Virus replication level test in duckling organs

[0221] The H5N6 subtype avian influenza virus S230 strain was used for challenge. On the third day after challenge, three SPF ducklings from each group were euthanized and dissected for analysis. The hearts, livers, spleens, lungs, kidneys, and brains of the SPF ducklings from each group were collected for plaque titration to determine the TCID of the H5N6 subtype avian influenza virus. 50 , as follows:

[0222] The hearts of SPF ducklings collected on the third day after infection were chopped into pieces and placed in a 2 mL EP tube. PBS containing 10,000 units of penicillin and streptomycin was added to the EP tube at a ratio of 1 g:1 mL, and high-pressure sterilized steel balls were placed in the tube. The tube was ground for 10 minutes using a low-temperature high-throughput tissue grinder, and then placed in a -80°C refrigerator and frozen and thawed three times. The tube was then centrifuged at 1000 rpm / min and 4°C for 10 minutes to collect the supernatant of the heart tissue of the SPF ducklings.

[0223] DF-1 cells were plated in a 96-well cell plate and cultured using a cell culture medium. When the DF-1 cells in each well grew to 80% of the well area, the cell culture medium was discarded to obtain a DF-1 cell plate to be used;

[0224] The heart tissue supernatant of SPF ducklings was diluted 10-fold (dilution 10 -1 ~10 8 ) after dilution, the heart tissue supernatant of SPF ducklings of each dilution was inoculated into DF-1 cell plates at 100 μL / well (the supernatant of each dilution was repeated in 6 wells), and the plates were placed in a cell culture incubator at 37°C and 5% (v / v) CO2 for incubation for 1 hour. After the incubation, the liquid in each well was discarded, and DMEM containing 2% (w / w) FBS was added at 100 μL / well, and the plates were placed in a cell culture incubator at 37°C and 5% (v / v) CO2 for incubation for 72 hours. After the incubation, the supernatant of each well was collected and the hemagglutination test (HA) was performed to determine the infection of the cells in each well with avian influenza virus, and the virus titer in the heart of the SPF ducklings was calculated based on the Reed-Muench method.

[0225] The liver, spleen, lung, kidney and brain of the SPF ducklings collected on the 3rd day after the infection were subjected to the same treatment to obtain the virus titer in the liver, spleen, lung, kidney and brain of the SPF ducklings on the 3rd day after the infection.

[0226] 7. Duckling serum conversion rate test

[0227] During the challenge with the H5N6 subtype avian influenza virus S230 strain, on the 14th day after the challenge, sera from 17 SPF ducklings in each group for evaluation and analysis were collected, and the hemagglutination inhibition test (HI) was performed according to the "WOAH Standard Manual".

[0228] 2. Experimental Results

[0229] 1. Antibody level test results

[0230] (1) IFN-γ and IL-4 level test results are shown in the figure Figure 12 As shown, A is the IFN-γ level test result graph, B is the IL-4 level test result graph, the results show that: the SPF ducklings in the negative control group had the average IFN-γ levels of 44.5 pg / mL, 47.2 pg / mL, 46.68 pg / mL and 43.3 pg / mL at 1 to 4 weeks after inoculation with DMEM cell culture medium, and the average IL-4 levels were 243.86 pg / mL, 253.67 pg / mL, 242.62 pg / mL and 228.84 pg / mL respectively; the SPF ducklings in the attenuated strain group had the nucleotide sequence obtained in Example 1 as shown in SEQ ID NO: 1 as shown in rS132-E SMT The average IFN-γ levels in the first to fourth weeks after inoculation were 70.89 pg / mL, 70.06 pg / mL, 89.8 pg / mL and 93.54 pg / mL, and the average IL-4 levels were 381.36 pg / mL, 469.15 pg / mL, 541.25 pg / mL and 666.72 pg / mL, respectively. The SPF ducklings in the live vector vaccine strain group were inoculated with the H5N6 subtype avian influenza live vector vaccine strain rS132-E obtained in Example 3, whose nucleotide sequence is shown in SEQ ID NO: 15. SMT The average IFN-γ levels in weeks 1 to 4 after -HAbNPt were 67.85 pg / mL, 73.28 pg / mL, 86.6 pg / mL and 96.9 pg / mL, respectively, and the average IL-4 levels were 391.33 pg / mL, 437.54 pg / mL, 550.33 pg / mL and 667.41 pg / mL, respectively.

[0231] The results show that the IFN-γ and IL-4 levels in the SPF ducklings of the attenuated strain group and the live carrier vaccine strain group are significantly higher than those in the SPF ducklings of the negative control group after immunization, indicating that the cellular immunity and humoral immunity of the SPF ducklings in the attenuated strain group and the live carrier vaccine strain group are induced and activated.

[0232] (2) The test results of the avian influenza virus neutralizing antibody titers of the SPF ducklings in each group are shown in the graph of Figure 13 The results show that the average titers of the neutralizing antibodies against the H5N6 subtype avian influenza virus in the SPF ducklings of the live carrier vaccine strain group are 1.39 log2, 1.78 log2, 2.68 log2 and 2.99 log2 respectively during the first to fourth weeks after immunization, while no neutralizing antibodies against the H5N6 subtype avian influenza virus are detected in the SPF ducklings of the negative control group and the attenuated strain group.

[0233] The results show that the H5N6 subtype avian influenza live carrier vaccine strain rS132-E obtained in Example 3 has the nucleotide sequence shown in SEQ ID NO: 15. SMT The H5N6 subtype avian influenza live carrier vaccine strain rS132-E obtained in Example 3 has the nucleotide sequence shown in SEQ ID NO: 15.

[0234] 2, Analysis results of the body weight of the ducklings

[0235] The analysis results of the body weight of the SPF ducklings in each group during the H5N6 subtype avian influenza virus S230 challenge are shown in the graph of Figure 14 The results show that the body weight of the SPF ducklings in the negative control group and the attenuated strain group slows down during the 8th to 10th day after challenge, and the body weight of the SPF ducklings in the negative control group increases by 1.67 times on the 14th day after challenge compared with that on the 0th day after challenge, and the body weight of the SPF ducklings in the attenuated strain group increases by 1.78 times on the 14th day after challenge compared with that on the 0th day after challenge.

[0236] The body weight of the SPF ducklings in the live carrier vaccine strain group steadily increases, and the body weight of the SPF ducklings in the live carrier vaccine strain group increases by 1.76 times on the 14th day after challenge compared with that on the 0th day after challenge.

[0237] 3, The survival rate and clinical symptom detection results of the ducklings

[0238] The survival rate results of the SPF ducklings in each group during the H5N6 subtype avian influenza virus S230 challenge are shown in the graph of Figure 15As shown in the figure, the results show that all SPF ducklings in the negative control group survived, 1 SPF duckling in the attenuated strain group died, the survival rate was 94.1%, and all SPF ducklings in the live carrier vaccine strain group survived, and the survival rate was 100%.

[0239] The figure of the detection results of the clinical symptoms of the SPF ducklings in each group during the H5N6 subtype avian influenza virus S230 challenge is as shown in Figure 16 As shown in the figure, A is the observation figure of the cornea of the SPF duckling, and B is the dissection observation figure of the SPF duckling.

[0240] The results show that on the 7th day after the SPF ducklings in the negative control group were challenged with the H5N6 subtype avian influenza virus S230, 5 SPF ducklings had decreased appetite and turbid cornea; on the 7th day after the SPF ducklings in the attenuated strain group were challenged with the H5N6 subtype avian influenza virus S230, 3 SPF ducklings had decreased appetite and turbid cornea, and 1 SPF duckling had obvious neurological damage symptoms (unstable standing), and the brain dissection figure is as shown in Figure 16 B, the brain appeared obvious hyperemia; and during the H5N6 subtype avian influenza virus S230 challenge, the SPF ducklings in the live carrier vaccine strain group did not have obvious clinical symptoms.

[0241] 4, Detoxification test results

[0242] The detoxification test results of the SPF ducklings in each group during the H5N6 subtype avian influenza virus S230 challenge (tested every 2 days after challenge) are shown in Table 7.

[0243] Table 7 Detoxification test results of SPF ducklings in each group after challenge

[0244]

[0245] Note: A is the negative control group, B is the attenuated strain group, and C is the live carrier vaccine strain group.

[0246] The results show that on the 2nd day after the SPF ducklings in the negative control group were challenged with the H5N6 subtype avian influenza virus S230, 1 SPF duckling had H5N6 subtype avian influenza virus shedding, on the 4th day after challenge, 1 SPF duckling had H5N6 subtype avian influenza virus shedding, on the 6th day after challenge, 6 SPF ducklings had H5N6 subtype avian influenza virus shedding, on the 8th day after challenge, 6 SPF ducklings had H5N6 subtype avian influenza virus shedding, on the 10th day after challenge, 1 SPF duckling had H5N6 subtype avian influenza virus shedding, and the total shedding rate was 52.9% (9 / 17).

[0247] In the attenuated strain group, two SPF ducklings showed H5N6 subtype avian influenza virus excretion on the second day after the infection with H5N6 subtype avian influenza virus S230, 10 SPF ducklings showed H5N6 subtype avian influenza virus excretion on the sixth day after the infection, 6 SPF ducklings showed H5N6 subtype avian influenza virus excretion on the eighth day after the infection, and 2 SPF ducklings showed H5N6 subtype avian influenza virus excretion on the tenth day after the infection. The total excretion rate was 64.7% (11 / 17).

[0248] In the live vector vaccine strain group, one SPF duckling showed H5N6 subtype avian influenza virus excretion on the 6th day after challenge with H5N6 subtype avian influenza virus S230, and five SPF ducklings showed H5N6 subtype avian influenza virus excretion on the 8th day after challenge (of which avian influenza virus was detected in both oropharyngeal and cloacal swabs of one duckling), with a total excretion rate of 29.4% (5 / 17).

[0249] The results show that the toxin excretion rate of the SPF ducklings in the live vector vaccine strain group was reduced by 23.5% compared with the toxin excretion rate of the SPF ducklings in the negative control group, and was reduced by 35.5% compared with the SPF ducklings in the attenuated strain group, indicating that the nucleotide sequence obtained in Example 3 is the H5N6 subtype avian influenza live vector vaccine strain rS132-E shown in SEQ ID NO: 15. SMT -HAbNPt immunization of ducklings can effectively reduce the damage caused by H5N6 subtype avian influenza virus to immunized SPF ducklings.

[0250] 5. Test results of virus replication levels in duckling organs

[0251] The results of the virus replication level test in the main organs of each group of SPF ducklings during the H5N6 subtype avian influenza virus S230 challenge period are shown in the figure. Figure 17 The results showed that after the SPF ducklings in the negative control group were challenged with H5N6 subtype avian influenza virus S230, avian influenza virus was detected in their heart, liver, spleen, lung, kidney, brain and trachea, with an average titer of 10 1.63 TCID 50 / 0.1mL, 10 3.53 TCID 50 / 0.1mL, 10 3.1 TCID 50 / 0.1mL, 10 4.33 TCID 50 / 0.1mL, 10 4.91 TCID 50 / 0.1mL, 10 4.98 TCID 50 / 0.1mL and 10 3.24 TCID 50 / 0.1mL; after the attenuated strain group of SPF ducklings were challenged with H5N6 subtype avian influenza virus S230, avian influenza virus was detected in the heart, liver, spleen, lung, kidney, brain and trachea, with an average titer of 10 1.63 TCID 50 / 0.1mL, 10 3.53 TCID 50 / 0.1mL, 10 3.1 TCID 50 / 0.1mL, 10 4.33 TCID 50 / 0.1mL, 10 4.91 TCID 50 / 0.1mL, 10 4.98 TCID 50 / 0.1mL and 10 3.24 TCID 50 / 0.1mL; after the SPF ducklings in the live vector vaccine strain group were challenged with H5N6 subtype avian influenza virus S230, avian influenza virus was detected in their hearts, livers, kidneys and trachea, with an average titer of 10 1.02 TCID 50 / 0.1mL, 10 0.77 TCID 50 / 0.1mL, 10 1.7 TCID 50 / 0.1mL and 10 0.88 TCID 50 / 0.1mL, and no avian influenza virus was detected in the spleen, lungs and brain.

[0252] The results show that after the SPF ducklings in the live vector vaccine strain group were infected with the H5N6 subtype avian influenza virus, the average titer of avian influenza virus in the heart was lower than the average titer of avian influenza virus in the heart of the SPF ducklings in the attenuated strain group and the negative control group, and the average titer of avian influenza virus in the liver, spleen, lung, kidney, brain and organs was significantly lower; this shows that the nucleotide sequence obtained in Example 3 is as shown in SEQ ID NO: 15. H5N6 live vector vaccine strain rS132-E SMT -HAbNPt can effectively reduce the damage caused by H5N6 subtype avian influenza virus to immunized SPF ducklings.

[0253] 6. Duckling serum conversion rate test results

[0254] The HI test results of each group of SPF ducklings during the H5N6 subtype avian influenza virus S230 challenge are shown in Table 8.

[0255] Table 8 HI test results of each group of SPF ducklings during the challenge period of H5N6 subtype avian influenza virus S230

[0256]

[0257] Note: A is the negative control group, B is the attenuated strain group, and C is the live vector vaccine strain group.

[0258] The results show that: the serum of 17 SPF ducklings for evaluation and analysis in the negative control group detected HI antibody titer on the 14th day after challenge, i.e. the serum conversion rate was 100% (17 / 17); the serum of SPF ducklings in the attenuated strain group detected HI antibody titer on the 14th day after challenge, i.e. the serum conversion rate was 100% (16 / 16, 1 died during challenge); the serum of 17 SPF ducklings for evaluation and analysis in the live vector vaccine strain group detected HI antibody titer on the 14th day after challenge, i.e. the serum conversion rate was 100% (17 / 17).

[0259] It is shown that the SPF ducklings in the negative control group, attenuated strain group and live vector vaccine strain group were successfully infected with H5N6 subtype avian influenza virus S230 strain, and the immune protection experiment results were established.

[0260] Finally, it should be explained that the above examples are only used to illustrate the technical solutions of the present application, but not to limit the protection scope of the present application. For those skilled in the art, on the basis of the above description and ideas, other different forms of changes or variations can also be made, which do not need or cannot be exhausted here. Any modification, equivalent replacement and improvement made within the spirit and principles of the present application shall be included in the protection scope of the claims of the present application.

Claims

1. An attenuated strain of duck Tembusu virus, characterized in that: The nucleotide sequence of the attenuated duck Tembusu virus strain is shown in SEQ ID NO:

1.

2. Use of the attenuated duck Tembusu virus strain according to claim 1 in the preparation of a vaccine for immunizing duck Tembusu virus.

3. Use of the attenuated duck Tembusu virus strain according to claim 1 in the preparation of a live vector vaccine strain; The live vector vaccine strain is a live vector vaccine strain targeting the H5N6 subtype avian influenza virus.

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