Mutation site for improving proliferation performance and immunogenicity of H9N2 subtype avian influenza virus and application

Abstract

The invention discloses a mutation site for improving proliferation performance and immunogenicity of an H9N2 subtype avian influenza virus and application, and belongs to the technical field of gene engineering. The mutation site is the 60th and / or 212th amino acid of the HA gene of the H9N2 subtype avian influenza virus strain. The recombinant virus is constructed by carrying out amino acid 60L and 212R mutation on an H9N2DSS isolate, and experiments show that the EID50 titer and virus copy number of the virus in a chick embryo can be improved by single-point mutation and combined mutation of 60L and 212R, the immunogenicity of the virus is improved, the induced antibody level and immune protection rate of the prepared inactivated vaccine are improved, and the inactivated vaccine has a good application prospect. Effective mutation sites are provided for preparing H9N2 subtype AIV vaccines which are high in immune protection rate and excellent in performance, and wide application prospects and practical value are achieved.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to a mutation site and its application for improving the proliferation performance and immunogenicity of H9N2 subtype avian influenza virus. Background Technology

[0002] Avian influenza (AI) is an infectious disease of birds caused by the avian influenza virus (AIV). The H9N2 subtype of AIV is a low-pathogenic avian influenza with a wide host range, including mammals. In current livestock and poultry farming practices, a transmission chain exists between poultry, pigs, and humans. Early symptoms of H9N2 subtype AIV infection include mild respiratory symptoms, decreased egg production (including soft-shelled and shell-less eggs), and reduced fertilization and feed conversion rates, but with a low mortality rate. H9N2 subtype AIV is prevalent globally and easily co-infects with infectious laryngotracheitis (ILT), Newcastle disease (ND), and infectious bronchitis (IB), and synergistically causes disease with Escherichia coli, resulting in significant losses to the poultry industry.

[0003] HA (Hypoxetine glycoprotein) is one of the two major surface glycoproteins of influenza virus, playing a crucial role in viral replication and antigenicity. HA is a primary target for neutralizing antibodies, inducing antibodies against HA in both vaccine-mediated and innate immunization. Several key amino acid positions in the H9 antigenic site have been identified as contributing factors to immune escape. Mutations at HA amino acid sites significantly affect viral antigenicity and host receptor binding properties, playing a vital role in viral replication and antigenicity. For example, they can enhance viral replication in chicken embryos and within chickens. Among the reported HA immune escape mutations, some have been found to have multiple functions. The study by Song et al. revealed the enhancing effect of the D200N mutation in the HA protein on viral replication. This amino acid mutation was particularly pronounced in chicken embryonic fibroblasts and chicken embryos. The D200N mutation in the HA1 region of the H9N2 subtype AIV added a glycosylation site at position 218, which not only altered the antigenic properties of the virus but also promoted viral replication in chicken embryos. In addition, the variation at position 145 of the glycosylation site also affected the replication ability of avian influenza virus in poultry. The N166D mutation not only altered the antigenicity of the virus but also affected the replication efficiency and pathogenicity of the virus in chickens.

[0004] Currently, the most widely used H9N2 subtype AIV vaccine in clinical practice remains the whole-virus inactivated vaccine. The preparation of the whole-virus inactivated vaccine and the viral load of the vaccine strain (EID) are crucial factors. 50 Closely related to the virus, increasing the viral titer in chicken embryos can effectively reduce the number of SPF chicken embryos required, thus lowering vaccine production costs. The HA protein is one of the two major surface glycoproteins of influenza viruses, playing a crucial role in viral replication and antigenicity. Amino acid mutations may affect protein expression and the replication ability of H9N2 subtype AIV. Site-directed mutagenesis of the HA protein amino acids of H9N2 subtype AIV, studying the effects of these mutations on the replication ability and immunogenicity of H9N2 subtype AIV in chicken embryos, and screening for amino acid mutations that can both improve viral replication and immunogenicity or have no effect on immunogenicity, is one effective way to improve the immunoprotective rate of H9N2 subtype AIV vaccines and reduce vaccine production costs. Summary of the Invention

[0005] To address the aforementioned technical problems, the present invention aims to provide a mutation site and its application for improving the proliferation performance and immunogenicity of H9N2 subtype avian influenza virus, thereby solving the problems of low antibody levels and low immune protection rates in H9N2 subtype AIV vaccines prepared from existing strains.

[0006] The technical solution of the present invention to solve the above-mentioned technical problems is as follows:

[0007] In a first aspect, the present invention provides a mutation site that enhances the proliferation performance and immunogenicity of H9N2 subtype avian influenza virus, wherein the mutation site is amino acid position 60 and / or position 212 of the HA gene of H9N2 subtype avian influenza virus strain.

[0008] Furthermore, the amino acid sequence of the HA gene is shown in SEQ ID NO.1;

[0009] SEQ ID NO.1:

[0010] DKICIGYQSTNSTETVDTLTENNVPVTHAKELLHTEHNGMLCATSLGQPLILDTCTIEGIIYGNPSCDLSLEGREWSYIVERPTAVHGLCYPGNVEDLEELRSLFSSARSYQRIQIFPDTIWNVSYDGTS TACSGSFYQSMRWLTRKNNEYPTQDAQYTNNQGKNILFMWGINHPPTDETQRGLYTRTDTTTSVATEEINRVFKPLIGPRPLVNGLMGRINYYWSVLKPGQTLRIKSDGNLIAPWYGHILSGESHGRILKT DLKRGSCTVQCQTEKGGLNTTLPFQNVSKYAFGNCSKYIGVKSLKLAVGLRNVPSRSSRGLFGAIAGFIEGGWPGLVAGWYGFQHSNDQGVGMAADRDSTQKAVDKITSKVNNIVDKMNKQYEIIDHEFN EVETRLNMINNKIDDQIQDIWAYNAELLVLLENQKTLDEHDANVNNLYNKVKRALGSNAVEDGKGCFELYHKCDDQCMETIRNGTYNRRKYQEESKLERQRIEGVKLESEGTYKILTIYSTVASSLVLAM.

[0011] In a second aspect, the present invention provides a method for improving the proliferation performance and immunogenicity of H9N2 subtype avian influenza virus, which involves site-directed mutation of amino acids at positions 60 and / or 212 of the HA gene of the H9N2 subtype avian influenza virus strain, followed by homologous recombination rescue and screening.

[0012] Furthermore, the amino acid sequence of the mutated HA gene is shown in any one of SEQ ID NO.2-SEQ ID NO.4;

[0013] SEQ ID NO.2: (I60L), the I at position 60 is mutated to L;

[0014] DKICIGYQSTNSTETVDTLTENNVPVTHAKELLHTEHNGMLCATSLGQPLILDTCTIEGLIYGNPSCDLSLEGREWSYIVERPTAVHGLCYPGNVEDLEELRSLFSSARSYQRIQIFPDTIWNVSYDGTSTACSGSFYQSMRWLTRKNNEYPTQDAQYTNNQGKNILFMWGINHPPTDETQRGLYTRTDTTTSVATEEINRVFKPLIGPRPLVNGLMGRINYYWSVLKPGQTLRIKSDGNLIAPWYGHILSGESHGRILKTDLKRGSCTVQCQTEKGGLNTTLPFQNVSKY AFGNCSKYIGVKSLKLAVGLRNVPSRSSRGLFGAIAGFIEGGWPGLVAGWYGFQHSNDQGVGMAADRDSTQKAVDKITSKVNNIVDKMNKQYEIIDHEFNEVETRLNMINNKIDDQIQDIWAYNAELLVLLENQKTLDEHDANVNNLYNKVKRALGSNAVEDGKGCFELYHKCDDQCMETIRNGTYNRRKYQEESKLERQRIEGVKLESEGTYKILTIYSTVASSLVLAM;

[0015] SEQ ID NO.3: (L212R), the L at position 212 is mutated to R;

[0016] DKICIGYQSTNSTETVDTLTENNVPVTHAKELLHTEHNGMLCATSLGQPLILDTCTIEGIIYGNPSCDLSLEGREWSYIVERPTAVHGLCYPGNVEDLEELRSLFSSARSYQRIQIFPDTIWNVSYDGTSTACSGSFYQSMRWLTRKNNEYPTQDAQYTNNQGKNILFMWGINHPPTDETQRGLYTRTDTTTSVATEEINRVFKPLIGPRPRVNGLMGRINYYWSVLKPGQTLRIKSDGNLIAPWYGHILSGESHGRILKTDLKRGSCTVQCQTEKGGLNTTLPFQNVSKYAFGNCSKYIGVKSLKLAVGLRNVPSRSSRGLFGAIAGFIEGGWPGLVAGWYGFQHSNDQGVGMAADRDSTQKAVDKITSKVNNIVDKMNKQYEIIDHEFNEVETRLNMINNKIDDQIQDIWAYNAELLVLLENQKTLDEHDANVNNLYNKVKRALGSNAVEDGKGCFELYHKCDDQCMETIRNGTYNRRKYQEESKLERQRIEGVKLESEGTYKILTIYSTVASSLVLAM;

[0017] SEQ ID NO.4: (I60L + L212R), where I at position 60 is mutated to L and L at position 212 is mutated to R;

[0018] DKICIGYQSTNSTETVDTLTENNVPVTHAKELLHTEHNGMLCATSLGQPLILDTCTIEGLIYGNPSCDLSLEGREWSYIVERPTAVHGLCYPGNVEDLEELRSLFSSARSYQRIQIFPDTIWNVSYDGTS TACSGSFYQSMRWLTRKNNEYPTQDAQYTNNQGKNILFMWGINHPPTDETQRGLYTRTDTTTSVATEEINRVFKPLIGPRPRVNGLMGRINYYWSVLKPGQTLRIKSDGNLIAPWYGHILSGESHGRILKT DLKRGSCTVQCQTEKGGLNTTLPFQNVSKYAFGNCSKYIGVKSLKLAVGLRNVPSRSSRGLFGAIAGFIEGGWPGLVAGWYGFQHSNDQGVGMAADRDSTQKAVDKITSKVNNIVDKMNKQYEIIDHEFN EVETRLNMINNKIDDQIQDIWAYNAELLVLLENQKTLDEHDANVNNLYNKVKRALGSNAVEDGKGCFELYHKCDDQCMETIRNGTYNRRKYQEESKLERQRIEGVKLESEGTYKILTIYSTVASSLVLAM.

[0019] A third aspect of the present invention provides the application of the above-described method for improving the proliferation performance and immunogenicity of H9N2 subtype avian influenza virus in the construction of H9N2 subtype avian influenza mutant strains.

[0020] In a fourth aspect, the present invention provides an H9N2 subtype avian influenza mutant strain, which is prepared by the above-described method for improving the proliferation performance and immunogenicity of the H9N2 subtype avian influenza virus.

[0021] A fifth aspect of the present invention provides the use of the above-mentioned mutation sites, methods, or H9N2 subtype avian influenza mutant strains in the preparation of H9N2 avian influenza inactivated vaccines.

[0022] The present invention has the following beneficial effects:

[0023] This invention constructs a recombinant virus by mutating amino acids 60L and 212R in the H9N2 DSS isolate. Experiments showed that both single-point and combined mutations of 60L and 212R can improve the EID (Enhanced Organic Intake) of the virus in chicken embryos. 50The mutation site can improve the titer and viral copy number, enhance the immunogenicity of the virus, increase the level of induced antibodies and the immune protection rate of the inactivated vaccine, and provide an effective mutation site for the preparation of H9N2 subtype AIV vaccines with high immune protection rate and superior performance. It has broad application prospects and practical value. Attached Figure Description

[0024] Figure 1 This is a comparison diagram of amino acid sites of HA in DSS and LJB isolates;

[0025] Figure 2 rDSS and its mutant strain chicken embryo EID 50 Proliferation curve;

[0026] Figure 3 For rJKR and its mutant strain chicken embryo EID 50 Proliferation curve;

[0027] Figure 4 The standard curve for quantitative PCR of pHW2000-M plasmid;

[0028] Figure 5 The graph shows the copy number proliferation curves of rDSS and its mutant strains of chicken embryo virus.

[0029] Figure 6 The graph shows the comparison of viral copy number in chicken embryos of rDSS and its mutant strains at different time points, where (a) is 36h, (b) is 24h, (c) is 48h, (d) is 60h, and (e) is 72h.

[0030] Figure 7 The graph shows the copy number proliferation curves of rJKR and its mutant strains in chicken embryo viruses.

[0031] Figure 8 The graph shows the comparison of viral copy numbers in chicken embryos of rJKR and its mutant strains at different time points, where (a) is 36h, (b) is 24h, (c) is 48h, (d) is 60h, and (e) is 72h.

[0032] Figure 9 This is a graph showing the virus copy number in the airway 5 days after the attack. Detailed Implementation

[0033] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0034] Example 1: Screening for mutation sites in the H9N2 HA gene

[0035] (1) Isolation, identification and propagation of H9N2 strain

[0036] Tracheal and lung tissue samples, as well as pharyngeal swabs, were collected from suspected diseased chickens. Sterile PBS containing 1000 U / mL each of penicillin and streptomycin was added at a 1:5 volume ratio. The mixture was ground five times in a grinder, followed by three freeze-thaw cycles. After centrifugation at 12000xg for 10 min, 0.2 mL of the supernatant was inoculated into 9-11 day old SPF chicken embryos through the allantoic cavity. The embryos were sealed with paraffin wax after inoculation. The embryos were observed daily, and embryos that died within 24 hours were discarded. After 72 hours of culture, the embryos were placed at 4℃ to shrink their blood vessels. Seven hours later, the allantoic fluid was collected for chicken erythrocyte hemagglutination assays, and nucleic acid was extracted for PCR identification. Ten H9N2 isolates were successfully isolated.

[0037] (2) HA experiment and EID 50 Measurement

[0038] Ten isolates were successfully isolated in step (1) and passaged. The HA hemagglutination titer of the allantoic fluid from chicken embryos collected from each generation was determined according to the "Diagnostic Techniques for Highly Pathogenic Avian Influenza GB / T18936-2020". The specific steps are as follows:

[0039] In a 96-well V-type microplate, add 25 μL of PBS to each well. Then, add 25 μL of virus solution to well 1 and mix by pipetting 3 to 5 times. Add 25 μL of virus solution from well 1 to well 2, mix by pipetting 3 to 5 times, and then add an equal amount of virus solution from well 2 to well 3. Continue this process until well 11, discarding an equal amount of virus solution from well 11. Well 12 is the PBS control well. After dilution, add 25 μL of PBS to each well. Then, add 25 μL of 1% chicken red blood cell suspension to each well and gently tap the plate to mix the reaction mixture. Let it stand at room temperature for about half an hour. When the PBS control wells show a distinct button-like appearance, observe the state of the red blood cells in each well, i.e., whether there is tear-like flow. The presence of tear-like flow indicates that the red blood cells have not agglutinated. When observing and judging, tilt the reaction plate at 60° and determine the hemagglutination titer as the highest dilution factor at which red blood cells completely agglutinate.

[0040] The virus was diluted tenfold to 10. 1 Up to 10 10 The diluted virus was then inoculated into 9-11 day old SPF chicken embryos, with 5 embryos per gradient. Each embryo was inoculated with 0.2 mL of virus solution and sealed with paraffin after inoculation. Embryo growth was observed daily, and embryos that died within 24 hours were discarded. After 72 hours of culture, the embryos were placed at 4°C to shrink their blood vessels. Seven hours later, allantoic fluid was collected for a hemagglutination assay using red blood cells, and the EID of the recombinant virus was calculated using the Reed-Munch method. 50The cells were passaged three times consecutively, and the experimental results are shown in Table 1.

[0041] Table 1. EID of chicken embryo passage of the virus strain 50

[0042]

[0043] As shown in Table 1, strains GDDSSH920221108 (DSS), FJHeyuanLJBH9202209 (LJB), and XNShilinJKRH920230201 (JKR) were able to be stably passaged in chicken embryos. Based on the results of the HA hemagglutination titer experiment, the DSS and LJB strains, which were able to be stably passaged in chicken embryos, were selected for amino acid site comparison. The JKR strain, which was located in a branch different from the DSS and LJB strains and was also able to be stably passaged in chicken embryos, was selected for auxiliary verification.

[0044] (3) HA protein amino acid comparison

[0045] Based on the results of the HA hemagglutination titer assay in step (2), strains that can be passaged in chicken embryos and have stable titers in each generation were screened out, and EID strains were selected from them. 50 Strains with titers below 7 and above 8 underwent amino acid comparison of the HA protein. Based on the amino acid comparison results, site-directed mutagenesis was performed on the selected sites.

[0046] Experimental results are as follows Figure 1 As shown, excluding the signal peptide, the DSS strain differs from the LJB strain at amino acid positions 60, 84, 139, 149, 202, and 226. Single-point mutations were performed at these six positions to construct recombinant plasmids, and subsequent experiments, including cell transfection and virus rescue, were conducted.

[0047] Example 2: Construction of recombinant virus rH9N2-DSS and its mutant virus

[0048] (1) PCR amplification and purification

[0049] The full-length gene fragments of HA, NA, PA, PB1, PB2, NP, M, and NS genes of JKR strain and DSS strain were amplified using the primers in Table 2. The amplification system is shown in Table 3. The amplified gene fragments contain BsmBI restriction sites.

[0050] Table 2 Primers for Avian Influenza Virus Whole Genome Amplification

[0051]

[0052] Table 3 PCR reaction system

[0053]

[0054] The amplification product was extracted using the Gel Extraction Kit (200) according to the instructions. The fragment was ligated into the pMD9-T vector according to the pMD19-T vector instructions. The ligation product was then transformed into DH5α competent cells. The specific steps were as follows: Competent cells taken from a -80°C freezer were placed on ice until almost thawed. 10 μL of the ligation product was added to the competent cells, and the bottom of the tube was gently tapped to mix. After incubating on ice for 30 min, the competent cells were heat-shocked in a 42°C water bath for 45 s, and immediately placed on ice to cool for 2-3 min. 500 μL of LB liquid medium was added, and the cells were incubated at 37°C for 1 h at 150 rpm. After incubation, the cells were centrifuged at 5000 rpm for 5 min, and 400 μL of supernatant was discarded. The remaining 100 μL of supernatant was gently resuspended by pipetting, and the bacterial culture was added to a preheated LA plate and gently mixed using a sterile spreader. The plates were incubated at 37°C for 16 hours. Single colonies were picked and identified by PCR using pMD19-T universal sequencing primers. Finally, five positive bacterial cultures for each gene fragment were sent to BGI Genomics Co., Ltd. in Guangzhou for sequencing.

[0055] (2) Construction of plasmids

[0056] Using the primers in Table 2, Max DNA Polymerase high-fidelity enzyme amplification was performed on the sample that was correctly sequenced in step (1). The reaction system was 50 μL, including 25 μL of... Max DNAPolymerase, 2 μL of upstream primer and 4 μL of downstream primer and bacterial culture template, with the remainder being RNase-free ddH2O; the reaction program was 98℃ for 2 min; 98℃ for 30 s, 55℃ for 15 s, 72℃ for 20 s, for 32 cycles; 72℃ for 10 min; store at 4℃.

[0057] The amplified target fragment was purified by gel extraction according to the instructions of the Gel Extraction Kit (200). After purification, the gene fragment was digested with restriction endonucleases. The BsmBI digestion reaction program was 55℃ for 6 h; the reaction was terminated at 80℃ for 20 min, and stored at 4℃. The digestion reaction system was 50 μL, which included 3 μL BsmBI, 12 μL of gel-extracted fragment, 5 μL rCutsmart, and 30 μL RNase-free ddH2O.

[0058] After gel recovery and enzyme digestion, the target gene fragment was ligated to the pHW2000 vector using T4 ligase. The ligation reaction system was 10 μL, including 1 μL T4 DNA Ligase, 1 μL DNA Ligase Buffer, and 8 μL gel-recovered fragment. Following step (1), the ligation product was transformed into DH5α competent cells. Colony PCR was performed using pHW2000 universal sequencing primers for identification. Five positive colonies for each gene fragment were selected and sent to BGI Genomics Co., Ltd. for sequencing.

[0059] (3) Plasmid extraction and sequencing

[0060] The bacterial culture with correct sequencing in step (2) was amplified and shaken. The amplified bacterial culture was then extracted using an endotoxin-free plasmid miniprep kit (BW-PD1212). After extraction, plasmids with A260 nm / A280 nm OD values ​​in the range of 1.80-2.0 were selected and sent to BGI Genomics Co., Ltd. in Guangzhou for sequencing. Plasmids with correct sequencing were selected for subsequent experiments.

[0061] Sequencing results showed that the present invention successfully constructed mutant plasmids rDSS-60, rDSS-84, rDSS-139, rDSS-149, rDSS-202 and rDSS-226.

[0062] (6) Plasmid transfection and virus rescue

[0063] Healthy 293T cells were seeded into 6-well plates and transfected when the cell density reached 70%-90%. Before the experiment, the cell culture medium in the 6-well plates was replaced with 800 μL of Opti-MEMI medium. The transfection complex was prepared by adding 16 μL of X-tremeGENE 9DNA Transfection Regent to 200 μL of Opti-MEMI medium and gently vortexing for 8 seconds. Then, rDSS pHW2000-HA and HA site-directed mutant plasmids, along with the corresponding strains pHW2000-NA, pHW2000-PA, pHW2000-PB1, pHW2000-PB2, pHW2000-NP, pHW2000-M, and pHW2000-NS plasmids (1 μg each) were added sequentially and gently vortexed for 8 seconds and incubated at room temperature for 25 minutes to obtain the transfection complex.

[0064] Next, the transfection complex was added dropwise to the cells, the 6-well plate was gently shaken to mix, and incubated at 37°C for 12 hours. After incubation, the 6-well plate was frozen and thawed twice at -80°C. The supernatant was then inoculated into 9-11 day old SPF chicken embryos, 0.4 mL per embryo. After incubation at 37°C for 72 hours, chicken embryos that died within 24 hours were discarded. The allantoic fluid from the chicken embryos was collected for HA hemagglutination titer determination, and the embryos were blindly passaged for 3 generations. If no HA hemagglutination titer was found, passage was continued until HA hemagglutination titer was obtained. Nucleic acid was extracted from the chicken embryo allantoic fluid, and the HA fragment was amplified using Hoffman influenza virus HA universal primers. The amplified product was sent to BGI Genomics Co., Ltd. in Guangzhou for sequencing. Chicken embryo allantoic fluid with correct sequencing results was selected for subsequent experiments.

[0065] Site-directed mutagenesis recombinant viruses were constructed at six sites. The rescue results showed that, compared to the rDSS control, only rDSS-60 effectively increased the viral hemagglutination titer by 2lg2 HA, and no mutations occurred after five consecutive passages. For other sites, titer was assessed after three passages, and all recombinant viruses after the third passage exhibited the 212R mutation. To investigate the effect of the 212R mutation, rDSS-212 and rDSS-60212 plasmids were constructed, transfected for rescue, and HA hemagglutination titer was measured. The results showed that both plasmids significantly increased the 3lg2 HA hemagglutination titer. Based on the experimental results, this invention screened recombinant viruses rDSS-60, rDSS-212, and rDSS-60212 with single and double site mutations at amino acids 60 and 212 for subsequent experiments. Based on the JKR strain, recombinant viruses rJKR-60, rJKR-212, and rJKR-60212 were constructed for subsequent experiments; the open reading frame amino acid sequences of the HA gene of the isolate DSS and the recombinant viruses rDSS-60, rDSS-212, and rDSS-60212 are shown in SEQ ID NO.1-SEQ ID NO.4, respectively.

[0066] Example 3: Analysis of the biological characteristics of recombinant viruses

[0067] (1) EID of recombinant virus 50 Measurement

[0068] First, the eight recombinant viruses obtained in Example 2, rDSS, rDSS-60, rDSS-212, rDSS-60212, rJKR, rJKR-60, rJKR-212, and rJKR-60212, were serially diluted 10-fold to obtain 10-10 recombinant viruses. 1 Up to 10 10The virus solution was then inoculated into 9-11 day old SPF chicken embryos. Five embryos were used for each gradient, and each embryo was inoculated with 0.2 mL of virus solution. The embryos were sealed with paraffin wax after inoculation. The embryos were observed daily by candling, and embryos that died within 24 hours were discarded. After 72 hours of culture, the embryos were placed at 4°C to allow for vasoconstriction. Allantoic fluid from the embryos was collected after the last 6 hours for a chicken erythrocyte hemagglutination assay. The EID of the recombinant virus was calculated using the Reed-Munch method. 50 .

[0069] (2) Recombinant virus in chicken embryo EID 50 Proliferation curve measurement

[0070] The proliferation curves of eight recombinant viruses on chicken embryos were determined at 24h, 36h, 48h, 60h, and 72h, with three chicken embryos used for each time period. All recombinant viruses were uniformly diluted to 200 EID. 50 Each recombinant virus was inoculated into 15 chicken embryos, and allantoic fluid from the embryos was collected at 24h, 36h, 48h, 60h, and 72h. The allantoic fluid collected at each time point was analyzed for viral load using the EID assay. 50 The measured data were then analyzed for significance using Graphpad Prism.

[0071] Experimental results are as follows Figure 2 and Figure 3 As shown, the EID measured in chicken embryo allantoic fluid collected at 60 h 50 Reaching peak levels. Compared to the unmutated recombinant virus rDSS, all mutant strains increased viral load by 10%. 0.5 -10 2 EID 50 EID after mutation at position 212 50 The increase was even more pronounced; combined mutations at positions 60 and 212 further increased viral load. In the rJKR experimental group, compared with the recombinant rJKR strain without any mutations, all mutant strains showed a 10% increase in viral load. 0.5 ~10 1.5 EID 50 Among them, the enhancement effect at position 212 was comparable to that at position 60. Similarly, combined mutations at positions 60 and 212 could further increase viral load. The results indicate that mutations at positions 60 and 212 of the HA gene can effectively increase the viral EID. 50 .

[0072] (3) Determination of chicken embryo copy number proliferation curve of recombinant virus

[0073] 1. Establishment of a quantitative fluorescence standard curve

[0074] Using pHW2000-M plasmid as a standard, the concentration of pHW2000-M plasmid was determined. A 10-fold serial dilution was performed using the elution buffer from the endotoxin-free plasmid mini-prep kit (BW-PD1212). 100 μL (original plasmid concentration) and 10 μL (presumably referring to further dilutions) of each plasmid were taken. -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 10 -8 10 -9 10 -10 Ten gradient concentrations of plasmid were used as templates, with three replicates for each gradient concentration, and a negative control was included. The reaction system was prepared according to the instructions of the Evo M-MLV one-step RT-qPCR (probe method) kit, and real-time quantitative RT-PCR was performed using a Xi'an Tianlong Gentier 96R real-time fluorescence quantitative PCR amplification instrument. The reaction was repeated three times to establish a standard curve.

[0075] The concentration of the plasmid standard was 330 ng / μL, the OD value of the plasmid at A260 nm / A280 nm was 1.86, and the copy number of the recombinant plasmid was 9.76 × 10¹⁰ copies / μL. Plotting the logarithm of the initial template amount on the x-axis and Ct value on the y-axis, the standard curve equation was obtained as Y = -3.153X + 43.493, with a correlation coefficient R₀. 2 =0.996, amplification efficiency En = 107.561%. The standard curve for plasmid standards is as follows: Figure 4 As shown.

[0076] 2. Determination of virus copy number proliferation curve in chicken embryos

[0077] Nucleic acid was extracted from chicken embryo allantoic fluid, and real-time quantitative RT-PCR was performed according to the instructions of the Evo M-MLV one-step RT-qPCR (probe method) kit. The viral copy number in the chicken embryo allantoic fluid was calculated using a pre-established standard curve. All recombinant viruses were diluted to a uniform 10⁷ in the chicken embryo allantoic fluid before inoculation into SPF chicken embryos. Allantoic fluid was collected at five time points: 24h, 36h, 48h, 60h, and 72h. Three chicken embryos were inoculated at each time point, with a total of 15 embryos inoculated at each time point. Viral nucleic acid was extracted from the collected allantoic fluid, and real-time quantitative RT-PCR was performed to calculate the viral copy number. The data were analyzed for significance using Graphpad Prism.

[0078] Experimental results are as follows Figures 5-8As shown. The viral copy number of the collected chicken embryo allantoic fluid reached its peak between 48 and 60 hours. In the rDSS experimental group, compared with the recombinant virus without any point mutations, all mutant strains increased the viral copy number by 5-25 times to that of the unmutated virus, with the mutation at site 212 showing the most significant increase. Furthermore, when the 60 and 212 sites were mutated together, it was associated with EID... 50 The results were consistent, indicating that viral copy number could be further increased. In the rJKR experimental group, compared with recombinant viruses without any point mutations, all mutant strains increased viral copy number by 6-12 times to that of unmutated viruses, with the increase at site 212 and site 60 being comparable. Figure 6 An analysis of the fold increase in viral copy number at different time points showed that the largest fold increase was observed at 48 hours in the DSS (Digital Subsystem for Viruses). Figure 7 In JKR, the increase was greatest at 36 hours. Figure 8 ).

[0079] Example 4: Cross-hemagglutination inhibition (HI) test and cross-serum neutralization test

[0080] (1) Preparation of inactivated vaccines

[0081] The isolated strains DSS, recombinant strains rDSS, rDSS-60, rDSS-212, and rDSS-60212 were propagated, and nucleic acids were extracted from the propagated allantoic fluid for RT-PCR amplification. The PCR products were sent to Guangzhou BGI Genomics Co., Ltd. for sequencing. After confirming the sequencing results, each allantoic fluid sample was inactivated by adding formaldehyde solution (the final volume concentration of formaldehyde in the allantoic fluid and formaldehyde mixture was 0.2%), mixed well, and placed in a shaker at 37℃ and 150 rpm for 24 hours. The inactivated strains were sterile according to the "Veterinary Pharmacopoeia of the People's Republic of China" (2015 edition). The inactivated allantoic fluid was inoculated into the allantoic cavity of 9-11 day old SPF chicken embryos, with 0.2 mL inoculated into each embryo. The inoculated chicken embryos were incubated in a 37℃ biochemical incubator for 48 hours, and blindly passaged for 3 generations. The allantoic fluid from each generation was collected to test for hemagglutination. If no hemagglutination occurred, it meant that the virus had been inactivated. Add Tween 80 to the qualified inactivated allantoic fluid to ensure a final volume concentration of 4%. Then, thoroughly mix the allantoic fluid and Tween 80. Next, weigh the compound adjuvant to achieve a volume ratio of 3:2 with the chicken embryo allantoic fluid mixture, and use a high-shear dispersion emulsifier to mix the mixture and the compound adjuvant. During emulsification, place the beaker in an ice bath to reduce heat generated by friction during stirring and prevent high temperatures from affecting the efficacy of the inactivated vaccine. Emulsification is complete when the emulsion drops float in water and remain cohesive. Centrifuge 1 mL of the emulsified inactivated vaccine at 12000 rpm for 3 minutes; if no water-oil separation occurs, emulsification is successful. Store the inactivated oil vaccine at 4°C in the dark.

[0082] (2) Preparation of single-factor serum

[0083] Eight-week-old SPF chickens were divided into five groups of three, and housed in isolators with free access to feed and water. Each group was immunized with a prepared inactivated vaccine via intramuscular injection, with 0.5 mL per chicken. Twenty-one days post-immunization, blood was collected from the chickens using a combination of cardiac and carotid artery sampling methods to maximize blood volume. The collected blood was incubated at 37°C for 1 hour, followed by centrifugation at 3000 rpm for 20 minutes. The supernatant was collected to obtain single-factor serum. The serum was aliquoted and stored at -80°C for subsequent crossover HI and crossover neutralization experiments.

[0084] (3) Cross-hemagglutination inhibition (HI) test

[0085] According to the "Diagnostic Techniques for Highly Pathogenic Avian Influenza GB / T18936-2020", a cross-HI experiment was performed. The hemagglutination titer (HA) was determined according to the method in Example 1, and 4HAU virus antigen was prepared based on the HA hemagglutination titer results. The HA hemagglutination titer divided by 4 gives the dilution factor of the 4HAU-containing antigen. The accuracy was verified according to the method in "Diagnostic Techniques for Highly Pathogenic Avian Influenza GB / T18936-2020". 4HAU antigen with correct test results was used for subsequent experiments. The test method is as follows: The prepared 4HAU antigen was diluted to a final dilution of 1:2 to 1:7 (7 dilutions in total). 25 μL was taken from each dilution, and an equal volume of PBS and then an equal volume of 1% chicken red blood cell suspension were added. After mixing, the mixture was allowed to stand at room temperature for 40 min. An agglutination endpoint appeared in the well at the 1:4 dilution, indicating that the prepared antigen solution was correct.

[0086] In a 96-well V-type microplate, 50 μL of PBS was added to each well except for the well in column 12, where 25 μL of PBS was added to each well. Then, 25 μL of serum was added to well 1 and mixed by pipetting 3 to 5 times. 25 μL of serum was then added to well 2 from well 1 and mixed by pipetting 3 to 5 times. Similarly, an equal amount of serum from well 2 was added to well 3, and so on, until well 11. 25 μL of serum was then discarded from well 11. Well 12 was the PBS control well. After dilution, 25 μL of 4HAU antigen was added to each well and incubated at room temperature for 30 min. Then, 25 μL of 1% chicken red blood cell suspension was added to each well. The reaction plate was gently tapped to mix the reaction mixture, and the plate was incubated at room temperature for 30 min. When the PBS control wells showed a distinct button-like appearance, the state of the red blood cells in each well was observed, i.e., whether tear-like flow was observed. During observation and determination, the reaction plate is tilted at 60°, and the highest serum dilution that completely inhibits the 4HAU antigen is used to determine the HI antibody titer of the serum. An HI antibody titer not higher than 1:8 (23 or 3 log2) is considered negative, and not lower than 1:16 (24 or 4 log2) is considered positive. The antigen correlation coefficient formula is then used for further analysis. Calculate the antigen correlation coefficient R, where r1 represents the HI titer of virus A against serum B / the HI titer of virus A against serum A, and r2 represents the HI titer of virus B against serum A / the HI titer of virus B against serum B.

[0087] The experimental results are shown in Tables 4 and 5.

[0088] Table 4. Cross-HI antibody titer

[0089]

[0090] Table 5 Antigen correlation coefficients in cross-HI experiments

[0091]

[0092] As shown in Table 4, the cross-HI antibody titers revealed a 0-16 fold difference between the strains. Table 5 shows that the antisera for rDSS and rDSS-60 were less protective than those for rDSS-212 and rDSS-60212 strains, indicating that mutations at amino acid sites 60L and 212R may alter the viral antigenicity. The antigenic correlation coefficients (R) in Table 4 ranged from 0.67 to 1.5, indicating no significant difference in antigenicity among the strains, and that the rDSS-212 and rDSS-60212 strains exhibited identical antigenicity.

[0093] (4) Cross-serum neutralization test

[0094] Utilizing the principle of antigen-antibody binding, positive serum is mixed with the detected virus and inoculated into the allantoic cavity of chicken embryos. The neutralizing antibody titer is calculated using the Reed-Munch method based on the hemagglutination properties of the allantoic fluid, thus determining the ability of the positive serum to neutralize the antigen. This allows for indirect prediction of the level of B-cell-mediated immune response induced by antigen stimulation. Compared to the cross-HI assay, this method yields more stable and reliable results. Serum neutralization assays are divided into α-method (dilution of virus, fixation of serum) and β-method (dilution of serum, fixation of virus). This study used the method of diluting serum to fix the virus for cross-serum neutralization experiments on chicken embryos.

[0095] The cross-serum neutralization experiment consisted of 16 groups, with each recombinant strain undergoing cross-serum reactions with others. A total of 6 dilution gradients were set up, with 4 chicken embryos per gradient. Before the experiment, the titers of recombinant strains rDSS, rDSS-60, rDSS-212, and rDSS-60212 were determined, and all strains were diluted to 200 EID. 50 / 0.1mL. Non-specific inhibitory factors were removed from the collected single-factor serum by incubating at 56℃ for 30 min. The treated serum was then serially diluted 2-fold with physiological saline to 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, and 1:512. The six dilutions (1:16, 1:32, 1:64, 1:128, 1:256, and 1:512) were used to dilute the recombinant strains rDSS, rDSS-60, rDSS-212, and rDSS-60212 to 100 EID. 50 Take 0.1 mL and mix it with an equal volume of the treated single-factor serum. After mixing, incubate at 37℃ for 30 min. Inoculate each dilution of serum and virus mixture into four 10-day-old chicken embryos via the allantoic cavity, with a total inoculation of 0.2 mL per embryo. A positive control was also set up (each embryo inoculated with 0.1 mL of 100EID). 50 The virus was used as a pretreatment agent, and a blank control was prepared (each embryo was inoculated with 0.1 mL of 0.9% physiological saline). Inoculated chicken embryos were placed in a 37°C incubator and incubated for 24 hours. After incubation, embryos were observed by candling. Embryos that died within 24 hours were removed. The remaining embryos were incubated for 72 hours, and allantoic fluid was collected for HA hemagglutination assay. The neutralizing antibody titer was calculated using the Reed-Munch method. The antigen-antigen correlation coefficient was calculated and analyzed using the antigen-antigen correlation coefficient calculation formula.

[0096] The experimental results are shown in Tables 6 and 7.

[0097] Table 6. Cross-neutralizing antibody titers

[0098]

[0099] Table 7. Antigen correlation coefficients in cross-neutralization experiments

[0100]

[0101] According to the antigen correlation coefficient results in Table 7, there was no significant difference in antigenicity among the strains. The cross-HI antibody titers in Table 6 showed a 0-16 fold difference between strains, with rDSS antiserum exhibiting poor reactivity to other strains, especially those containing the 212R mutation. Compared to the unmutated recombinant virus, the cross-neutralizing antibody titers of the mutant virus antiserum were all increased. The cross-neutralizing antibody titers of rDSS-60, rDSS-212, and rDSS-60212 antiserums were all higher than those of rDSS. The improvement was more pronounced in recombinant strains containing the 212R mutation. The protective effects of rDSS-212 and rDSS-60212 antiserums against rDSS and rDSS-60 were better than those against their own antiserums, indicating that the 60L and 212R mutations at amino acid sites have a positive impact on the production of neutralizing antibodies in these strains.

[0102] Example 5: Cross-immune protection experiment

[0103] (1) Pathogenicity test

[0104] One hundred and two hundred 21-day-old SPF chickens were randomly divided into six groups: groups 1-5 were the experimental group and the cohabitation group, and group 6 was the blank control group. The experimental group and the cohabitation group were placed in the same isolator with the same virus. Twelve chickens were in the direct challenge group and six were in the cohabitation group. The blank control group consisted of twelve chickens.

[0105] The experimental group underwent viral challenge with eye drops and nasal drops, with a viral challenge dose of 10. 6 EID 50 / 0.2mL / bird. On days 3, 5, and 7 post-challenge, cloacal and pharyngeal swabs were collected from each group of chickens. The allantoic cavity was inoculated into 9-day-old chicken embryos, with 3 embryos inoculated per swab. Embryos that died within 24 hours were discarded, and the allantoic fluid was collected after 72 hours for HA assay. On day 5 post-challenge, 3 chickens from each of the challenge and control groups were dissected, and tracheal and lung tissues were collected. Nucleic acid was extracted from the allantoic fluid of the chicken embryos, and RT-qPCR was performed to calculate the viral copy number.

[0106] The experimental results are shown in Table 8 and Figure 9 As shown.

[0107] Table 8. Virus shedding status of the challenge group and the cohabitation group.

[0108]

[0109] Five days after challenge, autopsy results showed that the chickens in the recombinant virus group had no obvious lesions, but the chickens in the wild-type virus DSS group had obvious caseous material and mucus in their trachea, and some chickens had mild unilateral air sacculitis.

[0110] As shown in Table 8, the recombinant virus group had a 60% swab positivity rate on days 3 and 5 post-challenge. The wild-type DSS group had a 100% swab positivity rate on days 3 and 5 post-challenge. The swab positivity rate decreased in all challenge groups by day 7. No viral shedding was detected in any of the cohabitation groups of the recombinant virus on days 3, 5, and 7 post-challenge. In contrast, the cohabitation group of the wild-type DSS had a 66.7% swab positivity rate on days 3 and 5 post-challenge, which decreased to 33.3% on day 7.

[0111] like Figure 9 As shown, quantitative fluorescence results indicated that no virus was detected in the lung tissue of any group after challenge, but it was detected in the tracheal tissue. Furthermore, the viral copy number in the tracheal tissue of the wild-type DSS group was higher than that of all recombinant virus groups, while there was no significant difference in viral copy number in the trachea among the recombinant virus groups.

[0112] (2) SPF Chicken Immunoprotection Experiment

[0113] Three hundred 21-day-old SPF chickens were randomly divided into 6 groups of 50 chickens each. Groups 1-5 were the immunization groups, immunized with an inactivated vaccine made from recombinant virus or wild-type virus strains; Group 6 served as the challenge control group and was not immunized. Each immunization group was subcutaneously immunized in the neck with 0.2 mL / bird of inactivated vaccine made from isolate DSS, recombinant strains rDSS, rDSS-60, rDSS-212, or rDSS-60212. Twenty-one days after immunization, blood was collected from the jugular vein and incubated at 37°C for 2 hours. The serum separated from the supernatant was aspirated to detect serum antibodies and calculate the antibody titer.

[0114] Chickens from each of the above immunized groups were randomly divided into 5 groups, with 10 chickens in each group; chickens from the challenge control group were also divided into 5 groups, with 10 chickens in each group. The virus was administered via eye drops and nasal drops at a dose of 10... 6 EID 50 Chickens were challenged with a 0.2 mL dose of virus. Each immunization group and each subgroup within the challenge control group were challenged with isolates DSS, recombinant strains rDSS, rDSS-60, rDSS-212, and rDSS-60212, respectively. On days 3 and 5 post-challenge, cloacal and pharyngeal swabs were collected and inoculated into 9-day-old chicken embryos, with three embryos inoculated per swab sample. 24 hours after inoculation, dead embryos were discarded, and the allantoic fluid titer collected at 72 hours was determined using the HA hemagglutination assay. An HA value greater than 22 was used as the criterion for non-protection against challenge.

[0115] The average antibody titers of immunized chickens are shown in Table 9. The number of antibodies induced by the recombinant strain was higher than that of the rDSS group. Among them, rDSS-212 and rDSS-60212 induced more antibodies in chickens, while rDSS induced fewer antibodies.

[0116] Table 9 Average antibody titer in immunized chickens

[0117]

[0118] Calculate the relative protection rate of a vaccine using the formula for relative vaccine protection rate:

[0119] The relative protection rate of a vaccine = [incidence (or mortality) in the control group - incidence (or mortality) in the experimental group] / [incidence (or mortality) in the control group].

[0120] The experimental results are shown in Tables 10 and 11. On day 3 after challenge and immunization, the rDSS vaccine group showed the lowest protection rate against wild-type DSS. Compared to the wild-type DSS vaccine group, other mutant strain vaccine groups showed higher protection rates against wild-type DSS. As shown in Tables 12 and 13, on day 5 after challenge and immunization, the vaccine protection rates of all groups improved, but the protection rates of the rDSS vaccine group and the wild-type DSS vaccine group against wild-type DSS remained low.

[0121] Table 10 Results of protection against infection on day 3

[0122]

[0123] Table 11 Relative protection rate of vaccine on day 3

[0124]

[0125] Table 12 Results of protection against cross-challenge on day 5

[0126]

[0127] Table 13 Relative protection rate of vaccine on day 5

[0128]

[0129] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A mutation site that enhances the replication performance and immunogenicity of H9N2 subtype avian influenza virus, characterized in that, The mutation site is the 60th and / or 212th amino acid of the HA gene of the H9N2 subtype avian influenza virus strain.

2. The mutation site for improving the replication performance and immunogenicity of H9N2 subtype avian influenza virus according to claim 1, characterized in that, The open reading frame amino acid sequence of the HA gene is shown in SEQ ID NO.

1.

3. A method for improving the replication performance and immunogenicity of H9N2 subtype avian influenza virus, characterized in that, The HA gene of the H9N2 subtype avian influenza virus strain described in claim 1 or 2 is subjected to site-directed mutation, followed by homologous recombination rescue and screening.

4. The method for improving the proliferation performance and immunogenicity of H9N2 subtype avian influenza virus according to claim 3, characterized in that, The open reading frame amino acid sequence of the HA gene after mutation is shown in any one of SEQ ID NO.2-SEQ ID NO.

4.

5. The method for improving the replication performance and immunogenicity of H9N2 subtype avian influenza virus as described in claim 3 or 4 is used in the construction of H9N2 subtype avian influenza mutant strains.

6. An H9N2 subtype avian influenza mutant strain, characterized in that, It was prepared using the method described in claim 3 or 4 for improving the proliferation performance and immunogenicity of H9N2 subtype avian influenza virus.

7. The use of the mutation site described in claim 1 or 2, the method described in claim 3 or 4, or the H9N2 subtype avian influenza mutant strain described in claim 6 in the preparation of an inactivated H9N2 avian influenza vaccine.

Images