Influenza A virus mutant strain and application thereof in preparation of vaccine

Abstract

The invention relates to the field of genetic engineering and biological medicine, in particular to an influenza A virus mutant strain and application thereof in preparation of vaccines, and the influenza A virus mutant strain is obtained by mutation of acetylation modification sites on hemagglutinin protein of influenza A H1N1 subtype viruses. The influenza A virus mutant strain can reduce the pathogenicity of the virus to mice without reducing the antigenicity of the virus, so that the pathogenicity of the virus to the mice through intrapulmonary inoculation is remarkably reduced, meanwhile, pathological changes of virus infection are not generated, but complete challenge protection can be provided for the mice, and therefore, the influenza A virus mutant strain can be used for preparing vaccines.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and biomedicine, and in particular to a mutant strain of influenza A virus and its use in vaccine preparation. Background Technology

[0002] Influenza A virus belongs to the Orthomyxoviridae family. Its genome is an 8-segment single-stranded negative-sense RNA. The virus is mainly spherical, with some being filamentous, and has an envelope. The influenza A virus genome encodes more than 12 proteins, among which the main glycoproteins on the envelope surface are hemagglutinin (HA) and neuraminidase (NA). Based on the serotypes of HA and NA, influenza A virus is further divided into 18 HA subtypes and 11 NA subtypes. The replication of the influenza A virus genome is catalyzed by RNA-dependent RNA polymerase. Due to the lack of proofreading activity of RNA polymerase, RNA replication fidelity is low, making the influenza virus genome highly susceptible to mutation during replication. The accumulation of these mutations causes changes in the antigenicity of the influenza virus, known as antigenic drift. At the same time, the segmented nature of the influenza virus genome also makes it prone to rearrangement, resulting in antigenic shift. When a new influenza variant emerges and the population generally lacks protective antibodies against it, it leads to an influenza pandemic.

[0003] Influenza viruses are one of the most important pathogens threatening public health. The influenza viruses that primarily infect humans and pigs are the H1N1, H1N2, and H3N2 subtypes. The viruses that primarily infect poultry are the H9N2 subtype, which can also occasionally infect humans, causing serious illness.

[0004] Currently, vaccines are the primary means of influenza virus prevention and control. The main immunogenic protein in influenza virus vaccines is the HA protein, which primarily induces the body to produce neutralizing antibodies against the HA protein. Commonly used vaccines in clinical practice include inactivated vaccines and live attenuated vaccines. Live attenuated vaccines have stronger antigenicity but carry the risk of viral virulence enhancement and recombination. Current strategies mainly involve truncating or deleting the NS1 protein of the influenza virus, thereby rendering the virus unable to replicate and reducing its pathogenicity.

[0005] The hemagglutination inhibition (HI) test is widely used to detect the titer of neutralizing antibodies against the influenza virus HA protein. It is a WHO-recommended effective tool for assessing the immunogenicity of influenza vaccines, conducting seroepidemiological studies, and analyzing the antigenicity of influenza viruses. The HI test method has been used for 70 years and offers advantages such as ease of operation, low cost, and high throughput. The HI test is based on the fact that the influenza virus HA protein binds to erythrocyte receptors and agglutinates erythrocytes, detecting the titer of antibodies in serum that block the agglutination of erythrocytes by the influenza virus HA protein. Summary of the Invention

[0006] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a mutant strain of influenza A virus and its use in vaccine preparation, in order to solve the problems in the prior art.

[0007] To achieve the above and other related objectives, the present invention provides a mutant strain of influenza A virus, which is obtained by mutating the acetylation modification site on the hemagglutinin (HA) protein of influenza A H1N1 subtype virus.

[0008] In some embodiments of the present invention, the amino acid sequence of the hemagglutinin protein of the H1N1 subtype influenza A virus is shown in SEQ ID NO.1.

[0009] In some embodiments of the present invention, the acetylation modification site is selected from any one or more sites in the amino acid sequence shown in SEQ ID NO.1: position 157, position 169, position 418, and position 459.

[0010] In some embodiments of the present invention, the mutation mode of the acetylation modification site is selected from any one or more of the following sites: K157R, K157Q, K169R, K169Q, K418R, K418Q, K459R, K459Q.

[0011] The present invention also provides an isolated polynucleotide comprising a polynucleotide sequence encoding the hemagglutinin protein of the influenza A virus mutant strain.

[0012] The present invention also provides a nucleic acid construct comprising the isolated polynucleotides described above.

[0013] The present invention also provides an engineered cell containing the aforementioned nucleic acid construct or the genome in which exogenous isolated polynucleotides are integrated.

[0014] In some embodiments of the present invention, the engineered cells are obtained by co-transforming host cells with the nucleic acid construct, a nucleic acid construct containing non-structural protein coding genes of influenza A virus mutant strains, and a nucleic acid construct containing structural protein coding genes of influenza A virus mutant strains NA, M1, M2, NP, PA, PB1, PB2, and NEP.

[0015] This invention also provides any of the following applications of the influenza A virus mutant strain, the isolated polynucleotide, the nucleic acid construct, or the engineered cells:

[0016] (1) Application in the immunogenicity evaluation of influenza A virus vaccine;

[0017] (2) Application in the detection of neutralizing antibody content in immune serum of influenza A virus;

[0018] (3) Application in the protective evaluation of influenza A virus vaccines;

[0019] (4) Application in the preparation of animal models of influenza A virus infection;

[0020] (5) Application in the screening or efficacy evaluation of drugs for the prevention and / or treatment of diseases caused by influenza A virus;

[0021] (6) Use in the preparation of antibodies for the prevention and / or treatment of diseases caused by influenza A virus;

[0022] (7) Application in the preparation of influenza A virus vaccines.

[0023] The present invention also provides a vaccine comprising the influenza A virus mutant strain.

[0024] The present invention also provides a method for preparing the vaccine, the method comprising: culturing the engineered cells, harvesting the culture supernatant, and amplifying the harvested culture supernatant to obtain the vaccine.

[0025] As described above, the influenza A virus mutant strain of the present invention and its use in vaccine preparation have the following beneficial effects: The present invention discovered that the hemagglutinin protein of the H1N1 subtype influenza virus has multiple acetylation sites, which have an important influence on the pathogenicity of the virus in mice. Mutating these acetylation sites one by one to deacetylation and simulated acetylation can reduce the pathogenicity of some viruses in mice, especially the deacetylation site at the K157 site, which does not reduce the antigenicity of the virus. In the present invention, the pathogenicity of the virus is reduced by deacetylation mutation at this site without reducing the antigenicity of the virus, thus significantly reducing the pathogenicity of the virus in mice via intrapulmonary inoculation, while not causing viral infection lesions, but providing complete protection against viral challenge in mice. Attached Figure Description

[0026] Figures 1-1 to 1-4 The image shown is a secondary peak diagram of the A / WSN / 1933 mass spectrometry of this invention.

[0027] Figure 2-1 The display shows the growth curve of a simulated acetylated virus strain.

[0028] Figure 2-2 The display shows the growth curve of a simulated deacetylated virus strain.

[0029] Figure 3-1The graph shows the changes in mouse body weight.

[0030] Figure 3-2 The graph shows the changes in mouse survival rate.

[0031] Figure 3-3 The figure shows the viral load in the lungs of mice.

[0032] Figure 3-4 The image shows the results of HE staining on a mouse lung pathological section under a microscope at 40x magnification.

[0033] Figure 3-5 The image shows the results of HE staining on a mouse lung pathological section under a microscope at 100x magnification.

[0034] Figure 3-6 The image shows the results of IHC staining (NP protein antibody) on a mouse lung pathological section.

[0035] Figure 4-1 The data shows the mouse's weight over 28 consecutive days.

[0036] Figure 4-2 The data shows the survival rate of mice over 28 consecutive days.

[0037] Figure 4-3 This is an anatomical diagram of mouse lung tissue.

[0038] Figure 4-4 The results show the antibody levels in mouse serum. Detailed Implementation

[0039] This invention provides a mutant strain of influenza A virus, which is obtained by mutating the acetylation modification site on the hemagglutinin (HA) protein of influenza A H1N1 subtype virus.

[0040] In some embodiments of the present invention, the amino acid sequence of the hemagglutinin protein of the H1N1 subtype influenza A virus is shown in SEQ ID NO.1.

[0041] In some embodiments of the present invention, the acetylation modification site is selected from any one or more sites in the amino acid sequence shown in SEQ ID NO.1: position 157, position 169, position 418, and position 459.

[0042] In some embodiments of the present invention, the mutation mode of the acetylation modification site is selected from any one or more of the following sites: K157R, K157Q, K169R, K169Q, K418R, K418Q, K459R, K459Q.

[0043] In some embodiments of the present invention, the influenza A virus mutant strain further includes structural proteins NA, M1, M2, NP, PA, PB1, PB2 and NEP, as well as the non-structural protein NS1. The amino acid sequences encoding these structural or non-structural proteins may be mutated or wild-type.

[0044] In one embodiment, the amino acid sequence of the NA protein of the influenza A virus mutant strain is shown in SEQ ID NO. 2.

[0045] In one embodiment, the amino acid sequence of the M protein of the influenza A virus mutant strain includes the sequences shown in SEQ ID NO. 3 and 4.

[0046] In one embodiment, the amino acid sequence of the NP protein of the influenza A virus mutant strain is shown in SEQ ID NO. 5.

[0047] In one embodiment, the amino acid sequence of the PA protein of the influenza A virus mutant strain is shown in SEQ ID NO. 6.

[0048] In one embodiment, the amino acid sequence of the PB1 protein of the influenza A virus mutant strain is shown in SEQ ID NO. 7.

[0049] In one embodiment, the amino acid sequence of the PB2 protein of the influenza A virus mutant strain is shown in SEQ ID NO. 8.

[0050] In one embodiment, the amino acid sequence of the NS protein of the influenza A virus mutant strain includes the sequences shown in SEQ ID NO. 9 and 10.

[0051] The present invention also provides an isolated polynucleotide comprising a polynucleotide sequence encoding the hemagglutinin protein of the influenza A virus mutant strain.

[0052] The polynucleotide can be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or artificially synthesized DNA. DNA can be single-stranded or double-stranded.

[0053] The polynucleotide encoding the hemagglutinin protein of the present invention can be prepared by any suitable technique known to those skilled in the art, such as, but not limited to, recombinant DNA technology, chemical synthesis, etc.

[0054] Based on the already disclosed amino acid sequence of the hemagglutinin protein, due to the degeneracy of the codon, those skilled in the art can obtain the nucleotide sequence of the isolated polynucleotide while keeping its encoding amino acid sequence unchanged. This is a conventional technique in the art.

[0055] In some embodiments of the present invention, the polynucleotide comprises a nucleotide sequence as shown in any of SEQ ID NO. 28 to 30.

[0056] The present invention also provides a nucleic acid construct comprising the isolated polynucleotides described above.

[0057] The term "nucleic acid construct" refers to an artificially constructed nucleic acid segment that can be introduced into target cells or tissues. The nucleic acid construct can be various expression vectors, which include a vector backbone, i.e., an empty vector and an expression framework.

[0058] There is no specific limitation on the type of expression vector. An expression vector is a nucleic acid molecule that allows the insertion of foreign nucleotides without disrupting its ability to replicate and / or integrate into the host cell. Expression vectors may include nucleic acid sequences that allow them to replicate in the host cell, such as origins of replication. Expression vectors may also include one or more selective marker genes and other genetic factors. An expression vector is a vector containing the necessary regulatory sequences to enable the transcription and translation of one or more inserted genes. Expression vectors are selected from eukaryotic expression vectors or prokaryotic expression vectors.

[0059] The prokaryotic expression vector was selected from Escherichia coli expression vector, Bacillus subtilis expression vector, or Streptomyces expression vector.

[0060] The eukaryotic expression vector is selected from yeast expression vectors, insect expression vectors, or mammalian expression vectors. Examples of mammalian expression vectors include pHW2000, pCMV, and pcDNA3.

[0061] The host cells are selected from eukaryotic or prokaryotic host cells. Eukaryotic host cells are selected from fungi such as yeast, insects, birds, plants, *C. elegans* or nematodes, or mammalian host cells. Examples of mammalian cells are COS cells, juvenile hamster kidney cells, mouse L cells, LNCaP cells, Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, African green monkey cells, CV1 cells, Vero or Hep-2 cells. Examples of prokaryotic host cells include bacterial cells, such as *Escherichia coli*, *Streptomyces*, *Salmonella typhi*, or mycobacteria.

[0062] Those skilled in the art can transfect the expression vector into host cells using methods well known in the art to obtain cells containing the gene encoding hemagglutinin protein.

[0063] The present invention also provides an engineered cell containing the aforementioned nucleic acid construct or the genome in which exogenous isolated polynucleotides are integrated.

[0064] In some embodiments of the present invention, the engineered cells are obtained by co-transforming host cells with the nucleic acid construct, a nucleic acid construct containing non-structural protein-coding genes of influenza A virus mutant strains, and a nucleic acid construct containing structural protein-coding genes of influenza A virus mutant strains NA, M1, M2, NP, PA, PB1, PB2, and NEP. The host cells are HEK293T cells.

[0065] This invention also provides any of the following applications of the influenza A virus mutant strain, the isolated polynucleotide, the nucleic acid construct, or the engineered cells:

[0066] (1) Application in the immunogenicity evaluation of influenza A virus vaccine;

[0067] (2) Application in the detection of neutralizing antibody content in immune serum of influenza A virus;

[0068] (3) Application in the protective evaluation of influenza A virus vaccines;

[0069] (4) Application in the preparation of animal models of influenza A virus infection;

[0070] (5) Application in the screening or efficacy evaluation of drugs for the prevention and / or treatment of diseases caused by influenza A virus;

[0071] (6) Use in the preparation of antibodies for the prevention and / or treatment of diseases caused by influenza A virus;

[0072] (7) Application in the preparation of influenza A virus vaccines.

[0073] In (1) above, the application is specifically as a standard detection strain for evaluating the immunogenicity of vaccines.

[0074] In (3) above, the application is specifically as an attack strain used for evaluating the protective efficacy of a vaccine.

[0075] In (4) above, the animal model is preferably a mouse model.

[0076] In the above (1)-(7), the influenza A virus is the influenza A H1N1 subtype virus.

[0077] The present invention also provides a vaccine comprising the influenza A virus mutant strain.

[0078] The vaccine described in this invention can be a whole-virus inactivated vaccine or a live attenuated vaccine.

[0079] The vaccine may also contain adjuvants, including but not limited to aluminum adjuvants.

[0080] The present invention also provides a method for preparing the vaccine, the method comprising: culturing the engineered cells, harvesting the culture supernatant, and amplifying the harvested culture supernatant to obtain the vaccine.

[0081] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0082] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0083] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0084] Example 1: Virus preparation, purification, and protein proteometry analysis

[0085] MDCK cells were used as cells for viral replication. After digestion with 0.25% trypsin, MDCK cells were injected with 2×10⁻⁶ cells. 6 Cells were seeded at 1000 mcg / mL in 6-well plates using DMEM medium containing 10% FBS and 1% penicillin-dextrose antibiotics. After overnight incubation in a CO2 incubator, influenza virus A / 1933 / WSN (commercial strain, purchased from Sinocare: A / WSN / 1933(H1N1)) was inoculated at an MOI of 0.001 the next day. The medium was then replaced with serum-free medium containing 1‰ TPCK trypsin and 1% penicillin-dextrose antibiotics. After 48 hours of incubation in a CO2 incubator, the MDCK cell status was checked. If a certain degree of cytopathic effect was observed but not all cells had died, the cell supernatant was collected, centrifuged for 5 minutes (1000 rpm), and aliquoted into 15 mL centrifuge tubes. The supernatant was then stored at -80°C.

[0086] Virus purification and viral protein profile analysis: The A / 1933 / WSN virus stock solution was enriched to 40 mL and centrifuged at 27,000 rpm for 3 h using an ultracentrifuge. The solid precipitate at the bottom was observed to be virus particles. The virus particles were resuspended in distilled water and the solution was sent for mass spectrometry analysis.

[0087] The results are as follows Figures 1-1 to 1-4 As shown, proteomic analysis revealed acetylation, a post-translational modification, on the influenza virus HA protein. The secondary mass spectrometry profile showed four distinct peaks indicating acetylation modification on the HA protein.

[0088] Example 2: Construction of an influenza virus reverse genetics system and rescue of recombinant influenza viruses containing acetylation site mutations in the HA gene.

[0089] Using pHW-2000 plasmid as the basic framework for plasmid construction, multiple segments (NA, PB, PA, M, NS, and NP) of wild-type HA protein and H1N1 subtype influenza virus 1933 / A / WSN were cloned. The amino acid sequences of each protein and the nucleotide sequences of the plasmid are shown below. Viral cDNA templates were cloned using polymerase chain reaction (PCR), with specific reaction conditions following the instructions for the enzymes used.

[0090] The amino acid sequence of wild-type HA protein (SEQ ID NO.1)

[0091] MKAKLLVLLYAFVATDADTICIGYHANNSTDTVDTIFEKNVAVTHSVNLLEDRHNGKLCKLKGIAPLQLGKCNIIGWLLGNPECDSLLPARSWSYIVETPNSENGACYPGDFIDYEELREQLSSVSSLERFEIFPKESSWPNHTFNGVTASCSHRGKSSFYRNLLWLTKKGDSYPKLTNSYVNNKGKEVLVLWGVHHPSSSDEQQSLYSNGNAYVSVASSNYNRRFTPEIAARPKVKDQHGRMNYYWTLLEPGDTIIFEATGNLIAPWYAFALSRGFESGIITSNASMHECNTKCQTPQGSINSNLPFQNIHPVTIGECPKYVRSTKLRMVTGLRNIPSIQYRGLFGAIAGFIEGGWTGMIDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSIIEKMNTQFTAVGKEFNNLEKRMENLNKKVDDGFLDIWTYNAELLVLLENGRTLDFHDLNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCDNECMESVRNGTYDYPKYSEESKLNREKIDGVKLESMGVYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI

[0092] Amino acid sequence of NA protein (SEQ ID NO.2)

[0093] MNPNQKIITIGSICMVVGIISLILQIGNIISIWISHSIQTGNQNHTGICNQGSITYKVVAGQDSTSVILTGNSSLCPIRGWAIHSKDNGIRIGSKGDVFVIREPFISCSHLECRTFFLTQGALLNDKHSRGTFKDRSPYRALMSCPVGEAPSPYNSRFESVAWSASACHDGVGWLTIGISGPDDGAVAVLKYNRIITETIKSWRKNILRTQESECTCVNGSCFTIMTDGPSDGLASYKIFKIEKGKVTKSIELNAPNSHYEECSCYPDTGKVMCVCRDNWHGSNRPWVSFDQNLDYKIGYICSGVFGDNPRPKDGTGSCGPVSADGANGVKGFSYKYGNGVWIGRTKSDSSRHGFEMIWDPNGWTETDSRFSMRQDVVAITNRSGYSGSFVQHPELTGLDCMRPCFWVELIRGLPEEDAIWTSGSIISFCGVNSDTVDWSWPDGAELPFTIDK

[0094] Amino acid sequence of M1 protein (SEQ ID NO.3)

[0095] MSLLTEVETYVLSIVPSGPLKAEIAQRLEDVFAGKNTDLEVLMEWLKTRPILSPLTKGILGFVFTLTVPSERGLQRRRFVQNALNGNGDPNNMDKAVKLYRKLKREITFHGAKEIALSYSAGALASCMGLIYNRMGAVTTEVAFGLVCATCEQIADSQHRSHRQMVTTTNPLIRHENRMVLASTTAKAMEQMAGSSEQAAEAMDIASQARQMVQAMRTIGTHPSSSAGLKDDLLENLQAYQKRMGVQMQRFK

[0096] Amino acid sequence of M2 protein (SEQ ID NO.4)

[0097] MSLLTEVETPIRNEWGCRCNDSSDPLVIAANIIGILHLILWILDRLFFKCIYRRFKYGLKR GPSTEGVPESMREEYRKEQQNAVDVDDGHFVNIELE

[0098] Amino acid sequence of NP protein (SEQ ID NO.5)

[0099] MATKGTKRSYEQMETDGERQNATEIRASVGKMIDGIGRFYIQMCTELKLSDYEGRLIQNSLTIERMVLSAFDERRNKYLEEHPSAGKDPKKTGGPIYRRVDGKWRRELILYDKEEIRRIWRQANNGDDATAGLTHMMIWHSNLNDATYQRTRALVRTGMDPRMCSLMQGSTLPRRSGAAGAAVKGVGTMVMELIRMIKRGINDRNFWRGENGRRTRIAYERMCNILKGKFQTAAQRTMVDQVRESRNPGNAEFEDLIFLARSALILRGSVAHKSCLPACVYGSAVASGYDFEREGYSLVGIDPFRLLQNSQVYSLIRPNENPAHKSQLVWMACHSAAFEDLRVSSFIRGTKVVPRGKLSTRGVQIASNENMETMESSTLELRSRYWAIRTRSGGNTNQQRASSGQISIQPTFSVQRNLPFDRPTIMAAFTGNTEGRTSDMRTEIIRLMESARPEDVSFQGRGVFELSDEKATSPIVPSFDMSNEGSYFFGDNAEEYDN

[0100] Amino acid sequence of PA protein (SEQ ID NO.6)

[0101] MERIKELRNLMSQSRTREILTKTTVDHMAIIKKYTSGRQEKNPALRMKWMMAMKYPITADKRITEMIPERNEQGQTLWSKMNDAGSDRVMVSPLAVTWWNRNGPVTSTVHYPKIYKTYFEKVERLKHGTFGPVHFRNQVKIRRRVDINPGHADLSAKEAQDVIMEVVFPNEVGARILTSESQLTTTKEKKEELQGCKISPLMVAYMLERELVRKTRFLPVAGGTSSVYIEVLHLTQGTCWEQMYTPGGEARNDDVDQSLIIAARNIVRRATVSADPLASLLEMCHSTQIGGIRMVNILRQNPTEEQAVDICKAAMGLRISSSFSFGGFTFKRTSGSSVKREEEVLTGNLQTLKIRVHEGYEEFTMVGRRATAILRKATRRLIQLIVSGRDEQSIAEAIIVAMVFSQEDCMIKAVRGDLNFVNRANQRLNPMHQLLRHFQKDAKALFQNWGIESIDNVMGMIGILPDMTPSTEMSMRGVRISKMGVDEYSSAEKIVVSIDRFLRVRDQRGNVLLSPEEVSETQGTEKLTITYSSSMMWEINGPESVLVNTYQWIIRNWETVKIQWSQNPTMLYNKMEFEPFQSLVPKAVRGQYSGFVRTLFQQMRDVLGTFDTAQIIKLLPFAAAPPKQSGMQFSSLTINVRGSGMRILVRGNSPIFNYNKTTKRLTVLGKDAGPLTEDPDEGTAGVESAVLRGFLILGKEDRRYGPALSINELSNLAKGEKANVLIGQGDVVLVMKRKRNSSILTDSQTATKRIRMAIN

[0102] Amino acid sequence of PB1 protein (SEQ ID NO.7)

[0103] MDVNPTLLFLKVPAQNAISTTFPYTGDPPYSHGTGTGYTMDTVNRTHQYSERGRWTTNTETGAPQLNPIDGPLPEDNEPSGYAQTDCVLEAMAFLEESHPGIFETSCLETMEVVQQTRVDKLTQGRQTYDWTLNRNQPAATALANTIEVFRSNGLTANESGRLIDFLKDVMESMNKEEMEITTHFQRKRRVRDNMTKKMVTQRTIGKRKQRLNKRSYLIRALTLNTMTKDAERGKLKRRAIATPGMQIRGFVYFVETLARSICEKLEQSGLPVGGNEKKAKLANVVRKMMTNSQDTEISFTITGDNTKWNENQNPRMFLAMITYITRNQPEWFRNVLSIAPIMFSNKMARLGKGYMFESKSIKIRTQIPAEMLASIDLKYFNDSTRKKIEKIRPLLIDGTASLSPGMMMGMFNMLSTVLGVSILNLGQKRHTKTTYWWDGLQSSDDFALIVNAPNHEGIQAGVNRFYRTCKLLGINMSKKKSYINRTGTFEFTSFFYRYGFVANFSMELPSFGVSGINESADMSIGVTVIKNNMINNDLGPATAQMALQLFIKDYRYTYRCHRGDTQIQTRRSFEIKKLWEQTHSKAGLLVSDGGPNLYNIRNLHIPEVCLKWELMDEDYQGRLCNPLNPFVNHKDIESVNNAVIMPAHGPAKNMEYDAVATTHSWIPKRNRSILNTSQRGILEDEQMYQKCCNLFEKFFPSSSYRRPVGISSMVEAMVSRARIDARIDFESGRIKKEEFTEIMKICSTIEELRRQK

[0104] Amino acid sequence of PB2 protein (SEQ ID NO.8)

[0105] MERIKELRNLMSQSRTREILTKTTVDHMAIIKKYTSGRQEKNPALRMKWMMAMKYPITADKRITEMIPERNEQGQTLWSKMNDAGSDRVMVSPLAVTWWNRNGPVTSTVHYPKIYKTYFEKVERLKHGTFGPVHFRNQVKIRRRVDINPGHADLSAKEAQDVIMEVVFPNEVGARILTSESQLTTTKEKKEELQGCKISPLMVAYMLERELVRKTRFLPVAGGTSSVYIEVLHLTQGTCWEQMYTPGGEARNDDVDQSLIIAARNIVRRATVSADPLASLLEMCHSTQIGGVRMVNILRQNPTEEQAVDICKAAMGLRISSSFSFGGFTFKRTSGSSVKREEEVLTGNLQTLKIRVHEGYEEFTMVGRRATAILRKATRRLIQLIVSGRDEQSIAEAIIVAMVFSQEDCMIKAVRGDLNFVNRANQRLNPMHQLLRHFQKDAKVLFQNWGIESIDNVMGMIGILPDMTPSTEMSMRGVRISKMGVDEYSSAEKIVVSIDRFLRVRDQRGNVLLSPEEVSETQGTEKLTITYSSSMMWEINGPESVLVNTYQWIIRNWETVKIQWSQNPTMLYNKMEFEPFQSLVPKAVRGQYSGFVRTLFQQMRDVLGTFDTAQIIKLLPFAAAPPKQSGMQFSSLTINVRGSGMRILVRGNSPVFNYNKTTKRLTVLGKDAGPLTEDPDEGTAGVESAVLRGFLILGKEDRRYGPALSINELSNLAKGEKANVLIGQGDVVLVMKRKRNSSILTDSQTATKRIRMAIN

[0106] Amino acid sequence of NS1 protein (SEQ ID NO.9)

[0107] MDPNTVSSFQVDCFLWHVRKRVADQELGDAPFLDRLRRDQKSLRGRGSTLGLDIETATRAGKQIVERILKEESDEALKMTMASVPASRYLTDMTLEEMSRHWFMLMPKQKVAGPLCIRMDQAIMDKNIILKANFSVIFDRLETLILLRAFTEEGTIVGEISPLPSLPGHTDEDVKNAVGVLIGGLEWNNNTVRVSETLQRFAWRSSNENGRPPLTPKQKRKMAGTIRSEV

[0108] Amino acid sequence of NS2 protein (SEQ ID NO.10)

[0109] Nucleotide sequence of plasmid IHA (SEQ ID NO.11): MDPNTVSSFQDILMRMSKMQLGSSSEDLNGIITQFESLKLYRDSLGEAVMRMGDLHSLQNRNGKWREQLGQKFEEIRWLIEEVRHRLKITENSFEQITFMQALQLLLEVEQEIRTFSFQL

[0110] ATGAAGGCAAAACTACTGGTCCTGTTATATGCATTTGTAGCTACAGATGCAGACACA

[0111] ATATGTATAGGCTACCATGCGAA

[0112] CAACTCAACCGACACTGTTGACACAATATTCGAGAAGAATGTGGCAGTGACACATT

[0113] CTGTTAACCTGCTCGAAGACAGAC

[0114] ACAACGGGAAACTATGTAAATTAAAAGGAATAGCCCCACTACAATTGGGGAAATGT

[0115] AACATCATCGGATGGCTCTTGGGA

[0116] AATCCAGAATGCGACTCACTGCTTCCAGCGAGATCATGGTCCTACATTGTAGAAAC

[0117] ACCAAACTCTGAGAATGGAGCATG

[0118] TTATCCAGGAGATTTCATCGACTATGAGGAACTGAGGGAGCAATTGAGCTCAGTATC

[0119] ATCATTAGAAAGATTCGAAATAT

[0120] TTCCCAAGGAAAGTTCATGGCCCAACCACACATTCAACGGAGTAACAGCATCATGC

[0121] TCCCATAGGGGAAAAAGCAGTTTT

[0122] TACAGAAATTTGCTATGGCTGACGAAGAAGGGGGATTCATACCCAAAGCTGACCAA

[0123] TTCCTATGTGAACAATAAAGGGAA

[0124] AGAAGTCCTTGTACTATGGGGTGTTCATCACCCGTCTAGCAGTGATGAGCAACAGA

[0125] GTCTCTATAGTAATGGAAATGCTT

[0126] ATGTCTCTGTAGCGTCTTCAAATTATAACAGGAGATTCACCCCGGAAATAGCTGCAA

[0127] GGCCCAAAGTAAAAGATCAACAT

[0128] GGGAGGATGAACTATTACTGGACCTTGCTAGAACCCGGAGACACAATAATATTTGA

[0129] GGCAACTGGTAATCTAATAGCACC

[0130] ATGGTATGCTTTCGCACTGAGTAGAGGGTTTGAGTCCGGCATCATCACCTCAAACGC

[0131] GTCAATGCATGAGTGTAACACGA

[0132] AGTGTCAAACACCCCAGGGATCTATAAACAGCAATCTCCCTTTCCAGAATATACACC

[0133] CAGTCACAATAGGAGAGTGCCCA

[0134] AAATATGTCAGGAGTACCAAATTGAGGATGGTTACAGGACTAAGAAACATCCCATC

[0135] CATTCAATACAGAGGTCTATTTGG

[0136] AGCCATTGCTGGTTTTATTGAGGGGGGATGGACTGGAATGATAGATGGATGGTATGG

[0137] TTATCATCATCAGAATGAACAGG

[0138] GATCAGGCTATGCAGCGGATCAAAAGCACACAGAATGCCATTAACGGGATTACA

[0139] AACAAGGTGAACTCTATTATCGAG

[0140] AAAATGAACACTCAATTCACAGCTGTGGGTAAAGAATTCAACAACTTAGAAAAAA

[0141] GGATGGAAAATTTAAATAAAAAAGT

[0142] TGATGATGGGTTTCTGGACATTTGGACATATAATGCAGAATTGTTAGTTCTACTGGAA

[0143] AATGGAAGAACTTTGGATTTCC

[0144] ATGACTTAAATGTGAAGAATCTGTACGAGAAAGTAAAAAGCCAATTAAAGAATAAT

[0145] GCCAAAGAAATCGGAAATGGGTGT

[0146] TTTGAGTTCTACCACAAGTGTGACAATGAATGCATGGAAAGTGTAAGAAATGGGAC

[0147] TTATGATTATCCAAAATATTCAGA

[0148] AGAATCAAAGTTGAACAGGGAAAAGATAGATGGAGTGAAATTGGAATCAATGGGG

[0149] GTGTATCAGATTCTGGCGATCTACT

[0150] CAACTGTCGCCAGTTCACTGGTGCTTTTGGTCTCCCTGGGGGCAATCAGTTTCTGGA

[0151] TGTGTTCTAATGGGTCTTTGCAG

[0152] TGCAGAATATGCATCTGA

[0153] The framework of plasmids NA, PB1, PB2, PA, M, NS, and NP is the same as that of plasmid HA. The following are the sources of the coding sequences for the NA, PB, PA, M, NS, and NP fragments in the plasmids:

[0154] PB1: GenBank: CY034138.1

[0155] PB2: GenBank: CY034139.1

[0156] PA: GenBank: CY034137.1

[0157] NP: GenBank: CY034135.1

[0158] NA: GenBank: CY034134.1

[0159] M: GenBank: D10598.1

[0160] NS: GenBank: CY034136.1.

[0161] After polymerase chain reaction (PCR), nucleic acid gel electrophoresis was performed to verify band size. Using a gel extraction kit, the recovered cells were digested and ligated, then chemically transformed into *E. coli* DH5α competent cells. The competent cells were then cultured and amplified in LB broth without antibiotics for 1 hour, centrifuged at 5000 rpm, and plated onto ampicillin-resistant LB agar plates. After incubation at 37°C for 12-16 hours, cells were picked for PCR and sequencing to confirm the cloning results. Once *E. coli* containing the correct plasmid were cultured, approximately 10 mL of LB broth containing antibiotics was cultured and amplified for 12-16 hours, centrifuged at 5000 rpm, and the bottom colonies were collected. The plasmid was extracted using a plasmid extraction kit.

[0162] Constructing an HA gene containing acetylation site modification mutations:

[0163] To convert acetylation sites in the HA protein to non-acetylation sites and simulate sustained acetylation, site-directed mutagenesis was used to mutate lysine (K) to arginine (R) to simulate deacetylated lysine (because R substitution prevents acetylation and retains a positive charge). Additionally, lysine (K) was mutated to glutamine (Q) to simulate sustained acetylation (due to similar steric hindrance). The primer sequences used for site-directed mutagenesis are as follows:

[0164] 157R

[0165] F:CTCCCATAGGGGAAGAAGCAGTTTTTACAG(SEQ ID NO.12)

[0166] R:CTTCCCCTATGGGAGCATGATACTGTTAC(SEQ ID NO.13)

[0167] 157Q

[0168] F:CTCCCATAGGGGACAAAGCAGTTTTTACAG(SEQ ID NO.14)

[0169] R:GTCCCCTATGGGAGCATGATACTGTTACTC(SEQ ID NO.15)

[0170] 169R

[0171] F:TGCTATGGCTGACGAGGAAGGGGGATTCATAC(SEQ ID NO.16)

[0172] R:CTCGTCAGCCATAGCAAATTTCTGTAAAA(SEQ ID NO.17)

[0173] 169Q

[0174] F:TTGCTATGGCTGACGCAGAAGGGGGATTCATA(SEQ ID NO.18)

[0175] R:GCGTCAGCCATAGCAAATTTCTGTAAAAAC(SEQ ID NO.19)

[0176] 418R

[0177] F:TCAACAACTTAGAAAGAAGGATGGAAAATTTA(SEQ ID NO.20)

[0178] R: CTTTCTAAGTTGTTGAATTCTTTACCCACAGC (SEQ ID NO.21)

[0179] 418Q

[0180] F: TCAACAACTTAGAACAAAGGATGGAAAATTTA (SEQ ID NO.22)

[0181] R: TTTCTAAGTTGTTGAATTCTTTACCCACAG (SEQ ID NO.23)

[0182] 459R

[0183] F: TGACTTAAATGTGAGGAATCTGTACGAGAA (SEQ ID NO.24)

[0184] R: CTCACATTTAAGTCATGGAAATCCAAAGTTC (SEQ ID NO.25)

[0185] 459Q

[0186] F: CATGACTTAAATGTGCAGAATCTGTACGAG (SEQ ID NO.26)

[0187] R: GCACATTTAAGTCATGGAAATCCAAAGTTC (SEQ ID NO.27)

[0188] Nucleotide sequence of HA - K157R gene (SEQ ID NO.28)

[0189] ATGAAGGCAAAACTACTGGTCCTGTTATATGCATTTGTAGCTACAGATGCAGACACAATATGT

[0190] ATAGGCTACCATGCGAA

[0191] CAACTCAACCGACACTGTTGACACAATATTCGAGAAGAATGTGGCAGTGACACATTCTGTTA

[0192] ACCTGCTCGAAGACAGAC

[0193] ACAACGGGAAACTATGTAAATTAAAAGGAATAGCCCCACTACAATTGGGGAAATGTAACATC

[0194] ATCGGATGGCTCTTGGGA

[0195] AATCCAGAATGCGACTCACTGCTTCCAGCGAGATCATGGTCCTACATTGTAGAAACACCAAA

[0196] CTCTGAGAATGGAGCATG

[0197] TTATCCAGGAGATTTCATCGACTATGAGGAACTGAGGGAGCAATTGAGCTCAGTATCATCATT

[0198] AGAAAGATTCGAAATAT

[0199] TTCCCAAGGAAAGTTCATGGCCCAACCACACATTCAACGGAGTAACAGCATCATGCTCCCAT

[0200] AGGGGAAGAAGCAGTTTT

[0201] TACAGAAATTTGCTATGGCTGACGCAGAAGGGGGATTCATACCCAAAGCTGACCAATTCCTA

[0202] TGTGAACAATAAAGGGAA

[0203] AGAAGTCCTTGTACTATGGGGTGTTCATCACCCGTCTAGCAGTGATGAGCAACAGAGTCTCT

[0204] ATAGTAATGGAAATGCTT

[0205] ATGTCTCTGTAGCGTCTTCAAATTATAACAGGAGATTCACCCCGGAAATAGCTGCAAGGCCC

[0206] AAAGTAAAAGATCAACAT

[0207] GGGAGGATGAACTATTACTGGACCTTGCTAGAACCCGGAGACACAATAATATTTGAGGCAAC

[0208] TGGTAATCTAATAGCACC

[0209] ATGGTATGCTTTCGCACTGAGTAGAGGGTTTGAGTCCGGCATCATCACCTCAAACGCGTCAA

[0210] TGCATGAGTGTTAACACGA

[0211] AGTGTCAAACACCCCAGGGATCTATAAACAGCAATCTCCCCTTTCCAGAATATACACCCCAGTC

[0212] ACAATAGGAGAGTGCCCA

[0213] AAATATGTCAGGAGTACCAAATTGAGGATGGTTACAGGACTAAGAAACATCCCATCCATTCA

[0214] ATACAGAGGTCTATTTGG

[0215] AGCCATTGCTGGTTTTATTGAGGGGGGATGGACTGGAATGATAGATGGATGGTATGGTTATCA

[0216] TCATCAGAATGAACAGG

[0217] GATCAGGCTATGCAGCGGATCAAAAGCACACAGAATGCCATTAACGGGATTACAAACAA

[0218] GGTGAACTCTATTATCGAG

[0219] AAAATGAACACTCAATTCACAGCTGTGGGTAAAGAATTCAACAACTTAGAAAAAAGGATGG

[0220] AAAATTTAAATAAAAAAGT

[0221] TGATGATGGGTTTCTGGACATTTGGACATATAATGCAGAATTGTTAGTTCTACTGGAAAATGG

[0222] AAGAACTTTGGATTTCC

[0223] ATGACTTAAATGTGAAGAATCTGTACGAGAAAGTAAAAAGCCAATTAAAGAATAATGCCAA

[0224] AGAAATCGGAAATGGGTGT

[0225] TTTGAGTTCTACCACAAGTGTGACAATGAATGCATGGAAAGTGTAAGAAATGGGACTTATGA

[0226] TTATCCAAAATATTCAGA

[0227] AGAATCAAAGTTGAACAGGGAAAAGATAGATGGAGTGAAATTGGAATCAATGGGGGTGTAT

[0228] CAGATTCTGGCGATCTACT

[0229] CAACTGTCGCCAGTTCACTGGTGCTTTTGGTCTCCCTGGGGGCAATCAGTTTCTGGATGTGT

[0230] TCTAATGGGTCTTTGCAG

[0231] TGCAGAATATGCATCTGA

[0232] Nucleotide sequence of HA - K169Q gene (SEQ ID NO.29)

[0233] ATGAAGGCAAAACTACTGGTCCTGTTATATGCATTTGTAGCTACAGATGCAGACACAATATGT

[0234] ATAGGCTACCATGCGAA

[0235] CAACTCAACCGACACTGTTGACACAATATTCGAGAAGAATGTGGCAGTGACACATTCTGTTA

[0236] ACCTGCTCGAAGACAGAC

[0237] ACAACGGGAAACTATGTAAATTAAAAGGAATAGCCCCACTACAATTGGGGAAATGTAACATC

[0238] ATCGGATGGCTCTTGGGA

[0239] AATCCAGAATGCGACTCACTGCTTCCAGCGAGATCATGGTCCTACATTGTAGAAACACCAAA

[0240] CTCTGAGAATGGAGCATG

[0241] TTATCCAGGAGATTTCATCGACTATGAGGAACTGAGGGAGCAATTGAGCTCAGTATCATCATT

[0242] AGAAAGATTCGAAATAT

[0243] TTCCCAAGGAAAGTTCATGGCCCAACCACACATTCAACGGAGTAACAGCATCATGCTCCCAT

[0244] AGGGGAAAAAGCAGTTTT

[0245] TACAGAAATTTGCTATGGCTGACGCAGAAGGGGGATTCATACCCAAAGCTGACCAATTCCTA

[0246] TGTGAACAATAAAGGGAA

[0247] AGAAGTCCTTGTACTATGGGGTGTTCATCACCCGTCTAGCAGTGATGAGCAACAGAGTCTCT

[0248] ATAGTAATGGAAATGCTT

[0249] ATGTCTCTGTAGCGTCTTCAAATTATAACAGGAGATTCACCCCGGAAATAGCTGCAAGGCCC

[0250] AAAGTAAAAGATCAACAT

[0251] GGGAGGATGAACTATTACTGGACCTTGCTAGAACCCGGAGACACAATAATATTTGAGGCAAC

[0252] TGGTAATCTAATAGCACC

[0253] ATGGTATGCTTTCGCACTGAGTAGAGGGTTTGAGTCCGGCATCATCACCTCAAACGCGTCAA

[0254] TGCATGAGTGTTAACACGA

[0255] AGTGTCAAACACCCCAGGGATCTATAAACAGCAATCTCCCCTTTCCAGAATATACACCCCAGTC

[0256] ACAATAGGAGAGTGCCCA

[0257] AAATATGTCAGGAGTACCAAATTGAGGATGGTTACAGGACTAAGAAACATCCCATCCATTCA

[0258] ATACAGAGGTCTATTTGG

[0259] AGCCATTGCTGGTTTTATTGAGGGGGGATGGACTGGAATGATAGATGGATGGTATGGTTATCA

[0260] TCATCAGAATGAACAGG

[0261] GATCAGGCTATGCAGCGGATCAAAAGCACACAGAATGCCATTAACGGGATTACAAACAA

[0262] GGTGAACTCTATTATCGAG

[0263] AAAATGAACACTCAATTCACAGCTGTGGGTAAAGAATTCAACAACTTAGAAAAAAGGATGG

[0264] AAAATTTAAATAAAAAAGT

[0265] TGATGATGGGTTTCTGGACATTTGGACATATAATGCAGAATTGTTAGTTCTACTGGAAAATGG

[0266] AAGAACTTTGGATTTCC

[0267] ATGACTTAAATGTGAAGAATCTGTACGAGAAAGTAAAAAGCCAATTAAAGAATAATGCCAA

[0268] AGAAATCGGAAATGGGTGT

[0269] TTTGAGTTCTACCACAAGTGTGACAATGAATGCATGGAAAGTGTAAGAAATGGGACTTATGA

[0270] TTATCCAAAATATTCAGA

[0271] AGAATCAAAGTTGAACAGGGAAAAGATAGATGGAGTGAAATTGGAATCAATGGGGGTGTAT

[0272] CAGATTCTGGCGATCTACT

[0273] CAACTGTCGCCAGTTCACTGGTGCTTTTGGTCTCCCTGGGGGCAATCAGTTTCTGGATGTGT

[0274] TCTAATGGGTCTTTGCAG

[0275] TGCAGAATATGCATCTGA

[0276] Nucleotide sequence of HA - K418R gene (SEQ ID NO.30)

[0277] ATGAAGGCAAAACTACTGGTCCTGTTATATGCATTTGTAGCTACAGATGCAGACACAATATGTATA

[0278] GGCTACCATGCGAA

[0279] CAACTCAACCGACACTGTTGACACAATATTCGAGAAGAATGTGGCAGTGACACATTCTGTTA

[0280] ACCTGCTCGAAGACAGAC

[0281] ACAACGGGAAACTATGTAAATTAAAAGGAATAGCCCCACTACAATTGGGGAAATGTAACATC

[0282] ATCGGATGGCTCTTGGGA

[0283] AATCCAGAATGCGACTCACTGCTTCCAGCGAGATCATGGTCCTACATTGTAGAAACACCAAA

[0284] CTCTGAGAATGGAGCATG

[0285] TTATCCAGGAGATTTCATCGACTATGAGGAACTGAGGGAGCAATTGAGCTCAGTATCATCATT

[0286] AGAAAGATTCGAAATAT

[0287] TTCCCAAGGAAAGTTCATGGCCCAACCACACATTCAACGGAGTAACAGCATCATGCTCCCAT

[0288] AGGGGAAAAAGCAGTTTT

[0289] TACAGAAATTTGCTATGGCTGACGAAGAAGGGGGATTCATACCCAAAGCTGACCAATTCCTA

[0290] TGTGAACAATAAAGGGAA

[0291] AGAAGTCCTTGTACTATGGGGTGTTCATCACCCGTCTAGCAGTGATGAGCAACAGAGTCTCT

[0292] ATAGTAATGGAAATGCTT

[0293] ATGTCTCTGTAGCGTCTTCAAATTATAACAGGAGATTCACCCCGGAAATAGCTGCAAGGCCC

[0294] AAAGTAAAAGATCAACAT

[0295] GGGAGGATGAACTATTACTGGACCTTGCTAGAACCCGGAGACACAATAATATTTGAGGCAAC

[0296] TGGTAATCTAATAGCACC

[0297] ATGGTATGCTTTCGCACTGAGTAGAGGGTTTGAGTCCGGCATCATCACCTCAAACGCGTCAA

[0298] TGCATGAGTGTAACACGA

[0299] AGTGTCAAACACCCCAGGGATCTATAAACAGCAATCTCCCCTTTCCAGAATATACACCCCAGTC

[0300] ACAATAGGAGAGTGCCCA

[0301] AAATATGTCAGGAGTACCAAATTGAGGATGGTTACAGGACTAAGAAACATCCCATCCATTCA

[0302] ATACAGAGGTCTATTTGG

[0303] AGCCATTGCTGGTTTTATTGAGGGGGGATGGACTGGAATGATAGATGGATGGTATGGTTATCA

[0304] TCATCAGAATGAACAGG

[0305] GATCAGGCTATGCAGCGGATCAAAAGCACACAGAATGCCATTAACGGGATTACAAACAA

[0306] GGTGAACTCTATTATCGAG

[0307] AAAATGAACACTCAATTCACAGCTGTGGGTAAAGAATTCAACAACTTAGAAAGAAGGATGG

[0308] AAAATTTAAATAAAAAAGT

[0309] TGATGATGGGTTTCTGGACATTTGGACATATAATGCAGAATTGTTAGTTCTACTGGAAAATGG

[0310] AAGAACTTTGGATTTCC

[0311] ATGACTTAAATGTGAAGAATCTGTACGAGAAAGTAAAAAGCCAATTAAAGAATAATGCCAA

[0312] AGAAATCGGAAATGGGTGT

[0313] TTTGAGTTCTACCACAAGTGTGACAATGAATGCATGGAAAGTGTAAGAAATGGGACTTATGA

[0314] TTATCCAAAATATTCAGA

[0315] AGAATCAAAGTTGAACAGGGAAAAGATAGATGGAGTGAAATTGGAATCAATGGGGGTGTAT

[0316] CAGATTCTGGCGATCTACT

[0317] CAACTGTCGCCAGTTCACTGGTGCTTTTGGTCTCCCTGGGGGCAATCAGTTTCTGGATGTGT

[0318] TCTAATGGGTCTTTGCAG

[0319] TGCAGAATATGCATCTGA

[0320] Saving the virus: Saving the HA gene containing acetylation site mutations, saving recombinant influenza viruses;

[0321] HEK 293T cells were plated and cultured overnight in an incubator. Under a microscope, the cells were observed to have reached 90%-100% confluency in the 6-well plates. Using an 8-plasmid reverse genetics system, the HA protein plasmid containing the mutation site and the remaining 7 segments were simultaneously co-transfected into HEK 293T cells. The transfection mass of each plasmid was at least 1 μg. After 48 hours, the cell status and cell culture medium color were observed. The cell supernatant was collected and used to infect MDCK cells. Cytopathic effect (CPE) was observed after 48 hours. If CPE occurred, the cell supernatant contained the virus successfully rescued by the reverse genetics system.

[0322] The collected cell supernatant was centrifuged at 1000 rpm to remove cell debris and then added to six-well plates containing MDCK cells. Serum-free viral culture medium containing 1‰ TPCK trypsin, 1% NEAA, and 1% penicillin antibiotics was used, and the plates were incubated in a CO2 incubator. MDCK cells were observed for cytopathic effects at 36 and 48 hours. Cytopathic effects were characterized by changes in cell morphology, clear cell borders, and the presence of dead cells floating on the surface. If cytopathic effects were observed, the cell supernatant was collected, centrifuged at 1000 rpm to remove cell debris, and the successfully rescued virus was stored at -80°C. The virus was passaged five times, and sequencing confirmed the absence of non-specific mutations.

[0323] TCID of successfully rescued mutant influenza virus strains 50 :

[0324] TCID 50Assay: MDCK cells were digested and placed in 96-well plates, incubated overnight in a CO2 incubator, and the cell status was observed the next day. The virus was removed from -80°C and thawed on ice beforehand, and serum-free medium containing 1‰ TPCK trypsin and 1% penicillin antibiotics was prepared. A new 96-well plate was prepared, and 135 μL of serum-free medium was added to each well. 15 μL of the original virus stock solution was added to each well in the first row, making the total volume of each well in the first row 150 μL, equivalent to a 10-fold dilution of the virus. The mixture was pipetted from the first row to the second row, and 15 μL of the mixture was transferred to the third row. This serial dilution process was repeated until the seventh row. The previously plated 96-well plate was then discarded. The remaining cell culture medium was washed with 100 μL of sterile PBS, and the PBS was discarded. The mixture with the lowest virus dilution titer was then transferred to the 96-well plate containing the cells. At this point, each row of the 96-well plate containing cells contained serially diluted virus solution. The plate was incubated in a CO2 incubator. Cytopathic effects were observed at 48 and 72 hours, and the virus dilutions at which cytopathic effects occurred were recorded. After 72 hours, the TCID of the 96-well plate was calculated using the Reed-Muench method. 50 The viral titers are shown in Table 1. The results indicate that the recombinant virus was successfully rescued and can replicate effectively in MDCK cells.

[0325] Table 1. Replication titers of the rescued recombinant virus on MDCK cells.

[0326] Virus <![CDATA[Titer Log2 TCID 50 > WSN-HA157R 5.2 WSN-HA157Q 6.5 WSN-HA169R 6.2 WSN-HA169Q 6.1 WSN-HA418R 6.7 WSN-HA418Q 7 WSN-HA459R 6.8 WSN-HA459Q 5.9 WSN-WT 6.8

[0327] Recombinant virus growth curve determination

[0328] MDCK cells were seeded in 12-well plates with an MOI of 0.001, and cell supernatants were collected at 12h, 24h, 36h, and 48h. The TCID values ​​of the cell supernatants collected at these four time points were then measured using MDCK cells. 50 The in vitro replication levels of influenza virus strains were obtained for the corresponding four time periods, such as Figure 2-1 and 2-2As shown in the figure. By analyzing the viral replication and growth curves at four time points, differences were found between the mutant strain and the original WSN strain. The growth and replication levels of strains with mutations at sites 157R, 169R, and 418R were lower than those of the wild-type virus, while the growth and replication capacity of the strain with the mutation at site 459R was similar to that of the wild-type virus. Among the simulated HA protein acetylation mutant viruses, the growth and replication level of the 418Q strain was significantly higher than that of the wild-type virus, while the in vitro growth and replication levels of 157Q, 169Q, and 459Q were slightly lower than or similar to those of WSN-WT. The in vitro growth and replication characteristics of the virus suggest that acetylation modification at sites K157, K169, and K418 of the HA protein in A / WSN / 1933 affects the in vitro growth and replication capacity of the virus. Deacetylation at these three sites downregulates the in vitro replication capacity of the virus, while acetylation at site K418 significantly increases the viral replication level at 24 h, and site K459 has no effect on the in vitro replication level of the virus.

[0329] Example 3: Pathogenicity experiment of recombinant virus in mice

[0330] Record the baseline weight of the mice one day in advance, and administer a nasal instillation of 10% diluted 10% thiocyanate solution to each mouse in each group. 4 TCID 50 The viral solution was diluted to a concentration of / mL and recorded as day 1 of inoculation. The mouse weight changes were continuously recorded for 14 days. The animal anesthetic, Sutacetin, was diluted 1:1 with sterile PBS. After weighing the mice, they were anesthetized by intramuscular injection into the right hind leg according to their weight in grams. The mice were anesthetized 5 minutes later. The mice were restrained and 20μL of viral solution was dripped into the nasal cavity of the mice using a pipette tip. The mice were carefully observed until they regained consciousness.

[0331] Twelve mice were divided into two cages and housed in each group. The cages were marked with different colored markers (numbers 1-6 and 7-12). To prevent the markings from fading over time, the markings were re-reinforced every 1-2 days. Mice were weighed and their weight recorded. Mice that lost more than 25% of their baseline weight or died during the observation period were considered dead. The survival rate after viral challenge was calculated.

[0332] Each group of mice was given 10 4 TCID 50 After challenge with / mL virus, necropsy was performed on the 3rd and 5th days post-challenge, with 3 mice randomly dissected each time, and lung tissue was taken from the mice to prepare tissue sections.

[0333] After collecting appropriately sized mouse lung tissue blocks, 1 mL of 4% paraformaldehyde was added for fixation, followed by paraffin mounting. Sections were prepared using the largest possible side and stained with hematoxylin and eosin (HE). Simultaneously, IHC staining was performed using the A / WSN / 1933 anti-NP protein antibody. HE staining was used to observe whether lung cells showed necrosis, edema, or inflammatory cell infiltration, and the pathological findings were used to assess the extent of the impact of different virus strains on mouse lungs. The distribution and intensity of the virus in the lungs were located using IHC staining with influenza virus NP protein antibody and HRP-labeled secondary antibody.

[0334] Mouse lung tissue was weighed and mixed with DMEM at a ratio of 0.1g lung tissue to 0.9mL. Steel balls were added for grinding, with each grinding cycle lasting 15 seconds, repeated three times until no tissue fragments were visible to the naked eye. The tissue was then centrifuged at 5000rpm to separate the supernatant and precipitate. The TCID value of the supernatant was determined. 50 .

[0335] The body weight and survival rate of mice were continuously recorded for 14 days after challenge. The results are as follows: Figure 3-1 , Figure 3-2 As shown, the weight changes in mice generally showed a trend of first decreasing and then increasing, except for the WSN-HA418Q strain, which experienced a sharp drop in weight and death on day 9. Regarding survival rates, compared to wild-type viruses, the WSN-HA157Q and WSN-HA418Q strains had lower survival rates, while the survival rates of WSN-HA169R, WSN-HA459R, and WSN-HA459Q were relatively higher, but still showed a trend towards weakened virulence. The remaining mutant viruses did not cause mouse mortality, achieving a survival rate of 100%.

[0336] Mouse weight changes and survival rates indicated that all HA protein mutant strains mimicking deacetylation exhibited significantly reduced replication capacity in animals, with some strains even failing to cause death compared to wild-type strains. However, the HA418Q and HA157Q strains mimicking HA protein acetylation caused significant weight changes and mortality rates in mice similar to wild-type viruses, suggesting that these two sites may be crucial for viral pathogenicity and have a significant impact on it. In conclusion, the mimicking persistent acetylation of HA protein K157 and K418 is important for viral pathogenicity in mice.

[0337] To determine the replication levels of WSN-WT and mutant viruses in the lungs of inoculated mice, each mouse was inoculated with 10^6 viruses. 4 TCID 50 / mL. Body weight changes and survival rate were recorded continuously for 14 days. Lung tissue was collected from mice on days 3 and 5 post-inoculation for processing, and the intrapulmonary titer after challenge was measured. Results are as follows: Figure 3-3 As shown.

[0338] From the changes in intrapulmonary titers in mice, except for the WSN-HA157R strain, the intrapulmonary titers in all mice showed a decreasing trend from 3 dpi to 5 dpi. Mice challenged with the HA-157R strain had higher intrapulmonary titers on day 5 than on day 3. This suggests that the HA protein-mimicking deacetylation modification at position 157 may have affected the disease progression in virus-infected animals, slowing the acute infection trend.

[0339] Three days after inoculation, the intrapulmonary titers of WSN-HA169R, WSN-HA459R, and WSN-HA418Q mice were significantly higher than those of the wild-type strain. The survival rates of WSN-HA169R and WSN-HA459R were consistent with the PBS group, with no deaths or significant weight changes, but their intrapulmonary titers were higher than those of the wild-type strain. WSN-HA418Q not only exhibited a higher viral load but also increased animal mortality. The intrapulmonary titers of WSN-HA459Q and WSN-HA157R were significantly lower than those of the wild-type virus. On day 5 after inoculation, the viral load of all mutant viruses was similar to that of the wild-type strain, with no significant difference. This suggests that acetylation at the K418 site enhances viral pathogenicity in mice and also increases its replication capacity in vivo, while deacetylation of K169 and K459, although increasing the replication capacity of the strain in the lungs to some extent, greatly reduces viral pathogenicity.

[0340] HE staining results are as follows Figure 3-4 and Figure 3-5 As shown, the lungs of mice in the PBS group did not show lesions. Under a 40x microscope, the lungs were smooth and without wrinkles, the parenchyma was covered by the outer membrane, the alveoli were evenly distributed, and there were irregular cavities in the cross-section of the trachea or bronchi, often accompanied by blood vessels. Under a 100x microscope, the alveolar septa in the PBS group were normal, the number of alveolar cells was normal, the alveolar wall thickness was normal, and there was no hemolysis or inflammatory cell infiltration. In contrast, in the lung pathological sections of the mutant virus group on the 3rd day after viral infection, HE staining under a 40x microscope revealed slight or obvious alveolar wall thickening, alveolar collapse, and a reduction or disappearance of alveolar septa. Some showed hemolysis, and various cells were scattered throughout. Under a 100x microscope, the lungs of the infected group showed erythrocyte and inflammatory cell infiltration, alveolar septal deformation, fewer and larger alveoli, fewer and deformed capillaries, and occasional multinucleated cells. Among them, WSN-HA418Q mice had severe hemorrhage or congestion in the alveoli of their lungs, while WSN-HA418R mice had severe inflammatory cell infiltration in their lungs.

[0341] IHC staining results are as follows Figure 3-6As shown, staining was performed using a specific NP antibody against the A / WSN / 1933 influenza virus, with the brown areas representing the stained viral regions. It is evident that some sections in the PBS group have a brown background, but the stained areas lack cell nuclei, which is due to background factors. The cells generally appear blue, without virus-specific staining. In contrast, the WSN-WT group and the mutant strain infection group showed virus-specific staining, indicating the distribution of the virus in the lungs. Overall, observation revealed that the viral distribution in the lungs of WSN-HA157R and WSN-HA459Q mice was significantly less than that in the WSN-WT group, while the lungs of WSN-HA418R and WSN-HA418Q mice showed dense brown staining, with numerous regional counts and widespread viral distribution. The viral distribution in sections from other strain-infected mice was similar to that in the WSN-WT group.

[0342] HE and IHC sections indicated that deacetylation at the K157 site significantly reduced viral replication in mouse lungs, and the viral titer in the lungs was also lower than that in WSN-WT. Meanwhile, the WSN-HAK418 site plays an important regulatory role in viral replication in mouse lungs. Although the viral distribution of WSN-HA418R and WSN-HA418Q was similar, the WSN-HA418R mouse group showed more severe inflammatory cell infiltration and alveolar wall thickening in the lungs, suggesting that deacetylation at the 418 site is more pathogenic in mice and can cause a more severe inflammatory response.

[0343] Example 4: Protective Experiment of Live Virus Vaccine

[0344] Previous mouse challenge experiments revealed that mice inoculated with the following strains—WSN-HA157R, WSN-HA169Q, WSN-HA418R, and WSN-HA459Q—had lower lung titers than the wild-type strain WSN, but higher survival rates. This suggests that the virulence of these four strains may be reduced in mice after mutation. This experiment aims to evaluate whether these mutated attenuated strains can serve as candidate strains for influenza virus vaccine preparation.

[0345] Four virus strains: WSN-HA157R, WSN-HA169Q, WSN-HA418R, and WSN-HA459Q, were tested at 10... 4 TCID 50 Mice were intranasally inoculated with a dose of / mL of the wild-type strain WSN, and their body weight and survival rate were recorded continuously for 14 days. Serum was collected from the mice on day 14, and they were subsequently intranasally inoculated again with a lethal dose of the wild-type strain WSN. 5 TCID 50 / mouse, record weight and survival rate, and study the changes in antibody levels in mice 14 and 28 days after challenge.

[0346] Sixty female BALB / c mice were randomly divided into five groups: WSN-HA157R, WSN-HA169Q, WSN-HA418R, WSN-HA459Q, and a PBS control group, with 12 mice in each group. Body weight and survival rate were recorded for 28 consecutive days.

[0347] After collecting blood from the posterior orbital plexus vein of mice, whole blood was incubated at room temperature for 1 hour, then overnight at 4°C. The next day, the blood was centrifuged at 5000 rpm for 10 minutes to obtain a layered liquid. The clear serum layer was collected into a new 1.5 mL centrifuge tube. The serum was treated to remove non-specific antibodies. 25 g of kaolin was dissolved in physiological saline and brought to a final volume of 100 mL, then stored at 2-8°C. 40 μL of serum from the mice to be tested was then added to 120 μL of the serum treatment solution prepared with kaolin. The mixture was shaken and incubated at room temperature for 30 minutes, then centrifuged at 3000 rpm for 10 minutes. The supernatant was collected into a new 1.5 mL centrifuge tube. 2 μL of chicken erythrocyte sediment was added to the supernatant, and the mixture was gently shaken and allowed to stand for 30 minutes. The centrifugation process was repeated (3000 rpm, 10 minutes). The supernatant at this point was the serum from the mice to be tested, diluted 4 times.

[0348] Mouse serum was collected 14 days after the first challenge and 14 days after the second challenge for hemagglutination inhibition experiments. First, 1% chicken erythrocytes were used to perform a hemagglutination assay to obtain the hemagglutination titer of the HA protein mutant virus strain. 25 μL of PBS was added to all wells of a microplate. 25 μL of each of the following virus suspensions (WSN-HA157R, WSN-169Q, WSN-HA418R, WSN-HA459Q, and A / 1933 / WSN) was added to well 1 and mixed thoroughly. Starting from well 1, 25 μL of the virus-PBS mixture was added to well 2 using a pipette, and this process was repeated until well 11. 25 μL of the mixture was then discarded from well 11. Next, 25 μL of 1% chicken erythrocyte suspension was added using a pipette, mixed thoroughly, and incubated at room temperature for 30 minutes. The effect was then observed; the erythrocytes in the control wells showed a distinct button-like shape, indicating the hemagglutination titer of each virus strain.

[0349] Prepare 4 units of antigen according to the titer of the hemagglutination assay, and perform a hemagglutination inhibition assay. Detect changes in serum antibody levels in mice 14 and 28 days after viral inoculation.

[0350] Fourteen days after the first viral challenge, mice were again intranasally inoculated with 10 [units of something]. 5 TCID 50A lethal dose of wild-type strain A / 1933 / WSN was administered per mL. Mice were dissected at 3 and 5 days post-mortem, and lung tissue was collected to determine the viral load of influenza virus. Mouse lung tissue was weighed, and appropriate culture medium and antibiotics were added at a ratio of 1 g: 9 mL DMEM. After tissue homogenization with steel beads, the tissue was centrifuged, and the supernatant and precipitate were separated. 15 μL of the supernatant was used to determine TCID. 50 .

[0351] Mouse body weight and survival rate were recorded continuously for 28 days, and the results are as follows: Figure 4-1 As shown in the diagram, consistent with previous animal experiments, the weight changes in mice reached their lowest point on days 8 and 9, followed by weight gain and stabilization. Using the PBS group as a control, after day 14, the mice's weight changes stabilized, demonstrating good tolerance to the lethal dose of WSN-WT without any deaths. Before day 14, except for the WSN-HA459Q group where mice died, other groups experienced some degree of weight loss, but no deaths occurred.

[0352] Three mice were randomly selected from each of the WSN-HA157R, WSN-HA169Q, WSN-HA418R, WSN-HA459Q, and PBS blank control groups. Lung tissue was dissected to observe for pathological changes such as necrosis, edema, and inflammatory exudation. The lung tissue... Figure 4-3 As shown. During lung tissue dissection, no obvious lesions were observed in the lungs of the challenged group compared to the control group. Cell culture medium was added according to the specified ratio, and the mixture was ground. After centrifugation, the supernatant was used to detect the viral load in the lungs. Results showed that no virus was detected in the lungs of mice with WSN-HA157R, WSN-HA169Q, WSN-HA418R, WSN-HA459Q, or the PBS control group. TCID levels were detected in MDCK cells. 50 The results showed that the cells were in good condition. No cellular pathological changes were observed with the naked eye.

[0353] The highest dilution of serum completely inhibiting the four-unit antigen was defined as the HI titer. A HI titer greater than or equal to 5 Log2 was considered positive. Based on the HI titers, at 14 dpi, the serum HI titers of mice in the WSN-HA157R group were all greater than or equal to 10 Log2; the serum HI titers of mice in the WSN-HA169Q group were discrete, but mainly concentrated at 9 Log2; the HI titers of mice in the WSN-HA418R group were mostly greater than 9 Log2; and in the WSN-HA459Q group, 2 mice had a titer of 9 Log2, 3 mice had a titer of 6 Log2, and 4 mice had a titer of 12 Log2. At 14 dpi, the WSN-HA157R group showed better immune response, with a relatively concentrated serum antibody titer greater than 5 Log2, indicating better protection. While the remaining three groups of mice also produced some immune responses, the immune response showed significant individual variability, with some mice having positive serum antibody titers but low HI levels. All mice inoculated with the virus had positive serum HI titers, which preliminarily indicates that inoculating mice with a weak strain of mutant HA protein can effectively stimulate an antiviral immune response and produce antibodies against this subtype of influenza virus.

[0354] The HI titer was obtained by performing a HI assay on the serum of mice at 14 days post-conception (dpc). The results are as follows: Figure 4-4 As shown, mice vaccinated with WSN-HA157R and WSN-HA169Q showed serum antibody titers reaching 12 Log2 at 14 days post-conversion (dpc). The antibody level in the WSN-HA157R group remained at a high HI titer, showing no significant difference between day 14 and day 14. However, the HI titer in the WSN-HA169Q group was significantly higher at day 14 compared to day 14 post-conversion (dpi), reaching 12 Log2. This suggests that re-challenging mice with a lethal dose of influenza virus can induce a higher antibody level, indicating that early immunization provides better protection. In contrast, the antibody titers in the WSN-HA418R and WSN-HA459Q groups, after re-challenging, fluctuated around 9 Log2, without the individual HI titer dispersion observed at day 14 post-conversion (dpi).

[0355] Based on the above data regarding mouse lung anatomy at 3 dpc and 5 dpc days, the measurement of viral load in the lungs, the serum antibody levels at 14 dpi and 14 dpc days, and the changes in mouse body weight over 28 consecutive days, it can be concluded that after vaccination with 10 4 TCID 50Mice inoculated with WSN-HA157R, WSN-HA169Q, and WSN-HA418R at concentrations of [volume value missing] mL exhibited good immunity and high antibody levels against lethal doses of the wild-type WSN strain. While mice inoculated with WSN-HA157R and WSN-HA169Q showed high antibody levels and strong immunity at 14 days post-vaccination, they also experienced significant weight fluctuations in the early stages. HA-418R, on the other hand, showed less weight change and higher safety; although its serum antibody titer at 14 days post-vaccination was only half that of the other two virus groups, it still achieved the required immunogenicity. Due to mouse mortality in the WSN-HA459Q group, further evaluation of its safety and reliability as a vaccine candidate strain may be necessary.

[0356] The above mouse immunization experiments showed that the antibody levels in the collected mouse serum were significantly higher than those in the PBS group. Some influenza virus strains with different degrees of acetylation modification of the HA protein can serve as potential influenza virus vaccine strains. Using the desired results as a reference, selecting appropriate mutant HA protein attenuated strains to prepare vaccines can provide good immune protection to the body.

[0357] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications and variations of the methods listed herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. A mutant strain of influenza A virus, characterized in that, The influenza A virus mutant strain was obtained by mutating the acetylation modification site on the hemagglutinin protein of the influenza A H1N1 subtype virus.

2. The influenza A virus mutant strain according to claim 1, characterized in that, The amino acid sequence of the hemagglutinin protein of the H1N1 subtype influenza A virus is shown in SEQ ID NO.

1.

3. The influenza A virus mutant strain according to claim 1, characterized in that, The acetylation modification site is selected from any one or more sites in the amino acid sequence shown in SEQ ID NO.1: position 157, position 169, position 418, and position 459.

4. The influenza A virus mutant strain according to claim 1, characterized in that, The mutation mode of the acetylation modification site is selected from any one or more of the following sites: K157R, K157Q, K169R, K169Q, K418R, K418Q, K459R, K459Q.

5. The influenza A virus mutant strain according to claim 1, characterized in that, The influenza A virus mutant strain also includes structural proteins NA, M1, M2, NP, PA, PB1, PB2, and NEP, as well as the non-structural protein NS1.

6. The influenza A virus mutant strain according to claim 5, characterized in that, It also includes one or more of the following features: 1) The amino acid sequence of the NA protein of the influenza A virus mutant strain is shown in SEQ ID NO.2; 2) The amino acid sequence of the M protein of the influenza A virus mutant strain includes the sequences shown in SEQ ID NO. 3 and 4; 3) The amino acid sequence of the NP protein of the influenza A virus mutant strain is shown in SEQ ID NO. 5; 4) The amino acid sequence of the PA protein of the influenza A virus mutant strain is shown in SEQ ID NO. 6; 5) The amino acid sequence of the PB1 protein of the influenza A virus mutant strain is shown in SEQ ID NO.7; 6) The amino acid sequence of the PB2 protein of the influenza A virus mutant strain is shown in SEQ ID NO. 8; 7) The amino acid sequence of the NS protein of the influenza A virus mutant strain includes the sequences shown in SEQ ID NO. 9 and 10.

7. An isolated polynucleotide, characterized in that, The isolated polynucleotides include polynucleotide sequences encoding the hemagglutinin protein of any of the influenza A virus mutants according to claims 1 to 6.

8. The isolated polynucleotide according to claim 7, characterized in that, The polynucleotide includes any of the nucleotide sequences shown in SEQ ID NO. 28 to 30.

9. A nucleic acid construct, characterized in that, The nucleic acid construct comprises the isolated polynucleotide as described in claim 7 or 8.

10. An engineered cell containing the nucleic acid construct of claim 9 or the genome of which is integrated an exogenous isolated polynucleotide of claim 7 or 8, the engineered cell further containing: a nucleic acid construct including a non-structural protein encoding gene of an influenza A virus mutant strain and a nucleic acid construct including structural protein encoding genes of influenza A virus mutant strains NA, M1, M2, NP, PA, PB1, PB2 and NEP.

11. Any of the following applications of the influenza A virus mutant strain according to any one of claims 1 to 6, the isolated polynucleotide according to claim 7 or 8, the nucleic acid construct according to claim 9, or the engineered cell according to claim 10: (1) Application in the immunogenicity evaluation of influenza A virus vaccine; (2) Application in the detection of neutralizing antibody content in immune serum of influenza A virus; (3) Application in the protective evaluation of influenza A virus vaccines; (4) Application in the preparation of animal models of influenza A virus infection; (5) Application in the screening or efficacy evaluation of drugs for the prevention and / or treatment of diseases caused by influenza A virus; (6) Use in the preparation of antibodies for the prevention and / or treatment of diseases caused by influenza A virus; (7) Application in the preparation of influenza A virus vaccines.

12. A vaccine, characterized in that, The vaccine includes any of the influenza A virus mutant strains described in claims 1 to 6.

13. The method for preparing the vaccine according to claim 12, characterized in that, The method includes: culturing the engineered cells as described in claim 10, harvesting the culture supernatant, and amplifying the harvested culture supernatant to obtain the vaccine.

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