WO2023036209A1 - 一种复制缺陷型耐药流感病毒及其核酸节段重组率检测方法 - Google Patents

一种复制缺陷型耐药流感病毒及其核酸节段重组率检测方法 Download PDF

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WO2023036209A1
WO2023036209A1 PCT/CN2022/117666 CN2022117666W WO2023036209A1 WO 2023036209 A1 WO2023036209 A1 WO 2023036209A1 CN 2022117666 W CN2022117666 W CN 2022117666W WO 2023036209 A1 WO2023036209 A1 WO 2023036209A1
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influenza virus
drug
protein
resistant
virus
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夏青
郑哲涛
史宁宁
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北京大学
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Definitions

  • the invention belongs to the field of biopharmaceuticals, and in particular relates to a drug-resistant replication-deficient influenza virus constructed by codon recoding technology, its application in treating drug-resistant influenza, and a method for detecting recombination rates of different segments of the virus.
  • a major mechanism of influenza A virus genetic diversity is the recombination of entire gene segments between co-infecting viruses. Recombination refers to the exchange of multiple single-stranded negative-sense RNA gene segments when multiple influenza viruses co-infect the same cell to form new progeny viruses.
  • the new strain after recombination may have changes in virus replication, pathogenic mechanism and host range. Due to the continuous recombination of influenza virus genes, seasonal influenza is constantly changing, and the difficulty of fighting influenza viruses is also increasing.
  • influenza virus can cause antigenic drift through genetic mutation, avoiding antibody-mediated antigen neutralization, causing the drug to lose its binding site with the virus, or the binding effect is reduced, resulting in the emergence of drug-resistant strains.
  • H274Y-NA mutation was the most common marker of oseltamivir resistance.
  • E119V mutation is most common and can lead to high levels of oseltamivir resistance.
  • sialic acid the natural substrate of NA protein
  • PDB 2BAT
  • Oseltamivir binds slowly to NA protein, but at the same time, due to the distorted side chain conformation, its binding is unstable, so it will appear slow binding and fast separation. Therefore, in order for the NA inhibitor to interact with the NA protein at this time, a higher concentration of the NA inhibitor is required, that is, the virus has developed drug resistance. Therefore, the recombination frequency of different gene segments of the virus is high, the frequency of viral recombination is high, and the virus is prone to drug resistance, which has become one of the technical problems to be solved in this field.
  • the mutation of influenza virus also brings challenges to the traditional multivalent inactivated influenza virus vaccine, leading to the uncertainty of immune effect.
  • Zhou Demin Zhang Lihe's team used reverse genetics technology to construct a non-replicating vaccine with comprehensive immune effects, transformed human embryonic kidney (HEK) 293T cells, and used lentiviral vectors to transfect an orthogonal prokaryotic Translation system, the modified 293T cell line is used for the packaging of influenza virus.
  • HEK human embryonic kidney
  • PTC premature termination codon
  • the research team finally selected a PTC-4A virus to study its safety, immunogenicity and protective effect.
  • the mutation of PTC-4A virus did not involve HA and NA genes, but involved PA, PB1, PB2 and NP.
  • This non-replicating virus can induce a comprehensive adaptive immune response, including high titers of hemagglutination inhibitory antibody (HI), neutralizing antibody (NT) and SIgA, and the number of virus-specific CTLs is 10 times higher than that of traditional vaccines.
  • the immune protection effect is similar to that of the current attenuated live vaccines, even better than the latter, especially for different serotypes of influenza viruses showing obvious cross-protection (Generation of influenza A viruses as live but replication-incompetent virus vaccines.Science, 354 (6316), 1170–1173.).
  • non-replicating influenza virus relies on the crossover of the antigen spectrum between the non-replicating influenza virus strain and the wild-type influenza virus circulating strain, and prevents influenza virus infection by triggering the body's immune response. Infections that do occur are less effective.
  • the non-replicating influenza virus can elicit a more comprehensive immune response through the way of endogenous antigen presentation, it has advantages in terms of cellular immune response compared with inactivated vaccines, but the antigen spectrum it provides is not as good as that of inactivated vaccines. richer.
  • anti-influenza virus drugs due to the wide application of anti-influenza virus drugs at present, while inhibiting the infection of influenza virus epidemic strains, it also inhibits the effect of vaccine strains.
  • the purpose of the present invention is to provide a brand-new antiviral strategy.
  • the drug-resistant virus can be constructed through codon recoding technology, gene mutation and molecular cloning technology.
  • Drug strain replication-deficient influenza virus using the natural characteristics of influenza virus segmentation and fragment recombination, can neutralize both wild-type influenza virus and drug-resistant influenza virus to achieve the effect of inhibiting proliferation, and verify the effect of NA Inhibitors can enhance the neutralization effect, and the drug-resistant influenza virus strain/drug combination NA28TAG/H274Y+oseltamivir with the best neutralization effect was screened out, and the virus strain+drug combination treatment was carried out at the mouse level for the first time Validation of the effect.
  • the invention provides a drug-resistant strain replication-deficient influenza virus constructed by using the characteristics of the influenza virus gene component segment and combining the gene codon expansion technology, and establishes a novel anti-influenza virus treatment method. Based on the genome sequences of known drug-resistant influenza virus strains, 12 drug-resistant influenza virus strains were obtained by molecular cloning technology, and their resistance rates to commonly used anti-influenza drugs were evaluated. Seven drug-resistant replication-deficient influenza virus strains (DRRIV) were constructed by introducing PTC mutations on the basis of drug-resistant influenza virus strains; a visual marker influenza virus for detecting the recombination rate of each segment of influenza virus was also constructed.
  • DRRIV drug-resistant replication-deficient influenza virus strains
  • NAIs small molecule NA inhibitors
  • the present invention provides a replication-deficient drug-resistant influenza virus (DRRIV), which is characterized in that the influenza virus produced by reverse genetics manipulation includes at least one drug-resistant mutation and at least one PTC mutation; wherein, The drug-resistant mutations are located in PA, NP, NA and/or M2 proteins, and the PTC mutations cause DRRIV to fail to proliferate and pass on in natural host cells; but in transgenic cells, when unnatural amino acids are added, the inserted The PTC loci are read through to achieve proliferation and passage.
  • DRRIV replication-deficient drug-resistant influenza virus
  • the replication-deficient drug-resistant influenza virus of the present invention is characterized in that the at least one drug-resistant mutation is located at an amino acid residue site selected from the group consisting of the 38th position of the PA protein, the 119th position of the PA protein, the NP Protein 289, NA protein 119, NA protein 274, NA protein 292, NA protein 294, M2 protein 26, M2 protein 27, M2 protein 31.
  • the numbering of amino acid residues of each protein of the replication-deficient drug-resistant influenza virus in the present invention is determined according to the A/WSN/33 (H1N1) strain.
  • the replication-defective drug-resistant influenza virus of the present invention is characterized in that the at least one drug-resistant mutation is selected from PA protein I38T, PA protein E119D, NP protein Y289H, NA protein E119V, NA protein H274Y, NA protein R292K , NA protein N294S, M2 protein L26F, M2 protein V27A, and M2 protein S31N.
  • any one of the aforementioned replication-deficient drug-resistant influenza viruses of the present invention is characterized in that the at least one drug-resistant mutation includes two drug-resistant mutations, preferably NA protein E119V+NA protein R292K, NA protein E119V+NA protein N294S , NA protein H274Y+NA protein N294S, NA protein H274Y+NA protein R292K, NA protein R292K+NA protein N294S.
  • the replication-defective drug-resistant influenza virus of the present invention is characterized in that the at least one PTC mutation is an insertion of a stop codon at an amino acid residue site selected from the group consisting of the 33rd position of the PB2 protein, the 33rd position of the PB1 protein 52, PA protein 266, HA protein 57, NP protein 101, NA protein 28, M2 protein 37, NS protein 131.
  • the replication-deficient drug-resistant influenza virus of the present invention is characterized in that the at least one PTC mutation is to mutate the original codon at the amino acid residue site to TAG.
  • the replication-deficient drug-resistant influenza virus of the present invention is characterized in that it includes a drug-resistant mutation to NA protein H274Y, a PTC mutation to a NA protein codon mutation at position 28 to TAG.
  • the present invention provides a visually marked influenza virus, which is characterized in that various structural proteins of influenza virus are marked by using three kinds of protein-specific labeling techniques; the three kinds of protein-specific labeling techniques use FIAsH-EDT2 to The four cysteine sequences (CCXXCC), the specific recognition of the 5 ⁇ Gly sequence by Sortase A enzyme, and the specific recognition of the ybbR sequence (DSLEFIASKLA) by Sfp synthetase were used for protein labeling.
  • the visually marked influenza virus of the present invention is characterized in that the tetracysteine sequence (CCXXCC) coding nucleic acid is inserted into the NP protein coding region and the M1 protein coding region, and the 5 ⁇ Gly sequence coding nucleic acid is inserted through reverse genetic manipulation HA protein coding region, insert ybbR sequence (DSLEFIASKLA) into NA protein coding region and M2 protein coding region.
  • CCXXCC tetracysteine sequence
  • 5 ⁇ Gly sequence coding nucleic acid is inserted through reverse genetic manipulation HA protein coding region, insert ybbR sequence (DSLEFIASKLA) into NA protein coding region and M2 protein coding region.
  • the present invention provides a method for detecting the recombination rate of influenza virus, which is characterized in that the host cell is co-infected with unlabeled influenza virus and any one of the aforementioned visually labeled influenza viruses, and the recombination is analyzed and calculated by detecting the ratio of the visualized signal of the recombinant progeny Rate.
  • the method for detecting the recombination rate of influenza virus according to the present invention is characterized in that the unlabeled influenza virus is the replication-deficient drug-resistant influenza virus described in any one of claims 1-7, and the recombination rate is encoded respectively Replication-deficient drug-resistant influenza virus genome segments of NP protein, M1 protein, HA protein, NA protein and M2 protein.
  • the present invention provides the genomic nucleic acid of any one of the aforementioned replication-deficient drug-resistant influenza viruses, which at least includes gene segments encoding NP, PB1, PB2, PA, M, NS, HA, and NA proteins, respectively.
  • the present invention provides a set of plasmids, including plasmids containing NP, PB1, PB2, PA, M, NS, HA, and NA protein expression cassettes respectively; said plasmid co-transfected host cells are capable of packaging and producing any of the aforementioned Replication-deficient drug-resistant influenza virus particles.
  • the present invention provides a composition comprising any of the aforementioned replication-deficient drug-resistant influenza viruses, the aforementioned genomic nucleic acid, or the aforementioned set of plasmids, and optional pharmaceutically acceptable carriers and/or Accessories.
  • composition of the present invention is characterized in that it also includes an effective amount of an influenza virus inhibitor.
  • influenza virus inhibitor is selected from the group consisting of NA inhibitors Oseltamivir, Zanamivir, PA inhibitors Baloxavir ), NP inhibitor Nucleozin, and derivatives thereof.
  • the present invention provides the application of any one of the aforementioned replication-deficient drug-resistant influenza viruses, the aforementioned genomic nucleic acid, the aforementioned set of plasmids, or any of the aforementioned compositions in the preparation of drugs for preventing and treating influenza virus infection.
  • the application of the present invention is characterized in that the drug is a vaccine; the influenza virus infection includes H1N1 subtype influenza virus infection.
  • influenza virus infection includes drug-resistant influenza virus infection and drug-non-resistant influenza virus infection.
  • the present invention provides the use of an influenza virus co-infection inducer in preparing and enhancing the neutralization activity of a replication-deficient drug-resistant influenza virus against influenza virus, characterized in that the replication-defective drug-resistant influenza virus is any one of the aforementioned Replication-deficient drug-resistant influenza virus.
  • influenza virus inhibitor of the present invention in the preparation and enhancement of the neutralization activity of replication-deficient drug-resistant influenza virus to influenza virus is characterized in that the influenza virus co-infection inducer is selected from the NA inhibitor oseltamivir (Oseltamivir), Zanamivir, PA inhibitor Baloxavir, NP inhibitor Nucleozin, and their derivatives.
  • influenza virus co-infection inducer is selected from the NA inhibitor oseltamivir (Oseltamivir), Zanamivir, PA inhibitor Baloxavir, NP inhibitor Nucleozin, and their derivatives.
  • the invention successfully constructed 12 drug-resistant influenza virus strains with different combinations for the first time, and the invention systematically evaluated the drug resistance rates of the 12 drug-resistant virus strains to different small molecule drug inhibitors.
  • seven different DRRIVs were successfully constructed by using codon recoding technology and molecular cloning technology.
  • the The virus genome containing PTC can be recombined into the progeny virus through fragment recombination, thereby inactivating (neutralizing) the wild-type virus, and the DRRIV virus has a good neutralizing effect on both the wild-type virus and the drug-resistant strain virus.
  • Different methods were used to systematically evaluate its neutralizing effect and broad-spectrum on drug-resistant viruses, and NA28TAG/H274Y drug-resistant virus strains were screened out.
  • influenza visualization viral protein labeling of transpeptidase
  • NAI NA inhibitor
  • FIG 1 Schematic diagram of drug resistance sites in different segments of influenza virus
  • PA, NP, NA, and M2 have different drug resistance sites and are sensitive to different drug resistance inhibitors.
  • PA drug-resistant influenza viruses can be successfully packaged
  • NA segment drug-resistant influenza viruses can be successfully packaged with one or two drug-resistant sites, and have obvious CPE effects, and three drug-resistant sites or more cannot be successfully packaged.
  • FIG. 3 The packaging and proliferation capabilities of 12 different drug-resistant influenza virus strains
  • Figure 3a Relative packaging efficiencies and viral titers of 12 different drug-resistant influenza virus strains
  • the inhibition rate of BAV on WSN-PA I38T drug-resistant strains was 8.486 ⁇ M, compared with that of the parent virus Compared with WSN, it has increased by about 18 times.
  • the NP-Y289H drug-resistant strain virus liquid was harvested, and the inhibition rate of the compound Nucleozin on the WSN-NP/Y289H drug-resistant strain was determined.
  • the inhibition rate of Nucleozin on the WSN-NP/Y289H drug-resistant strain was 20.08 ⁇ M, which was comparable to that of its parent strain WSN. Ratio increased by about 295 times.
  • Figure 5b Packaging efficiency and virus titer of DRRIV carrying drug-resistant mutation sites and PTC sites on transgenic cells supplemented with NAEK in the medium
  • DRRIV carrying drug-resistant mutation sites and PTC sites had no significant difference in the drug resistance rate and growth curve of the transgenic cells with NAEK added in the medium; DRRIV was unable to proliferate in NAEK.
  • Figure 6a The relative packaging efficiency and virus titer of DRRIV with higher packaging efficiency and virus titer in 7 kinds
  • DR-GRV-NA N28TAG+H274Y
  • PR8 and CA07 strains can neutralize not only WSN strains, but also PR8 and CA07 strains, and achieve broad-spectrum therapeutic effects within the H1N1 subtype.
  • it also has a neutralizing effect on the wild type of NA drug-resistant strains with mutations in other drug-resistant sites, indicating that it also has a neutralizing effect on other drug-resistant strains of influenza.
  • Figure 7b Neutralization of wild-type influenza, PA-I38T drug-resistant influenza, NP-Y280H drug-resistant influenza, and NA-H274Y drug-resistant influenza by PTC, PTC-PA-I38T, PTC-NP-Y280H, and PTC-NA-H274Y Activity (MOI 1:1).
  • FIG. 7c Neutralizing activity of DRRIV (NA-H274Y+NA-28TAG) against H1N1, drug-resistant H1N1, H3N2, influenza B viruses, CPE.
  • FIG. 8a Visually labeled influenza virus labeling scheme
  • FIG. 8b Visualization of labeled influenza viruses co-cultured with DRRIV to be neutralized.
  • the M2 and HA segments of the wild-type drug-resistant strain are marked, which are represented by green and red fluorescence, respectively.
  • the fluorescence intensity is significantly reduced, and the NA-H274Y+NA-28TAG mutation combined fluorescence The intensity is the lowest, which intuitively shows that DRRIV has a neutralizing effect on the wild-type drug-resistant influenza virus, and further verifies that the neutralizing effect of the NA-H274Y+NA-28TAG mutation combination is the best.
  • Fig. 9a-9f show that the low concentration of drugs of NAIs can greatly improve the neutralization ability of DRRIV virus (about 3 times), while neither PA inhibitor (BXM) nor NP inhibitor (Nucleozin) has this effect.
  • Oseltamivir has the most obvious promoting effect on DRRIV.
  • the influenza virus labeled with green fluorescence and the influenza virus labeled with red fluorescence to successively infect (interval 2h) cell carry out cell imaging at 12h, the cell can only be infected by the first kind of green fluorescent virus.
  • NAIs OSV and Zanamivir
  • were added green and red fluorescent viruses could be seen at the same time, while neither NPI nor PAI had this effect. Therefore, we concluded that NAIs can effectively promote the multi-infection ability of cells.
  • HA, NA, and M are similar methods, and the recombination rate is HA>NA>M.
  • NS, M, and NP are similar labeling methods, so the recombination rate is NS>M>NP.
  • the recombination efficiency of each segment in WSN influenza virus is HA>NA>M>NP.
  • b represents the survival rate of mice, in which the untreated group infected with DRV-NA all died on the seventh day, but the survival rate of mice in the DR-GRV-NA group was greatly increased, and the survival rate was 89%, while in the addition of DR-GRV -NA+OSV, the concentration of OSV is 1mg/kg, which is far lower than the effective concentration, and the survival rate is 100%.
  • c represents the body weight change of the mice after treatment, the body weight of the DR-GRV-NA treatment group and the DR-GRV-NA+OSV treatment group began to recover in 4-5 days, and the DR-GRV-NA+OSV treatment group recovered the body weight It is faster than the DR-GRV-NA treatment group, and it is proved from the animal level that the combination of DR-GRV-NA and low concentration of OSV can enhance the therapeutic effect of DR-GRV-NA.
  • d indicates that the mouse lung tissue and brain tissue were ground, the tissue RNA was extracted, and the viral load was detected by RT-qPCR.
  • the virus titer in the lung and brain decreased significantly, compared with the untreated group. groups have significant differences.
  • e and f represent the immunofluorescence detection of viral NP protein on frozen sections of lung tissue, the content of NP protein in the treatment group was significantly reduced, and at the same time, the results of HE staining on paraffin sections of lung tissue were consistent with the above.
  • the tissue-level verification verified that DR-GRV-NA can effectively treat DRV-NA.
  • Example 1 Construction of 12 Drug-resistant Influenza Virus Strains and Evaluation of Packaging Efficiency
  • virus maintenance medium DMEM+1% fetal bovine serum+2 ⁇ g/mL TPCK-trypsin
  • the TRIzol lysate in the above cells was transferred to a 1.5 mL centrifuge tube, and left at room temperature (15-30° C.) for 5 min.
  • add chloroform according to the ratio of 0.2mL chloroform per 1mL TRIzol close the cap of the centrifuge tube, shake vigorously for 15s, let stand at room temperature (15-30°C) for 3min, and centrifuge at 12000g (2-8°C). 15min.
  • RNA quantification was performed with a Nano-300 UV spectrophotometer. At the same time, the growth curve was measured, and the cells were subcultured to a 6-well plate, 10 6 /well, and cultured at 37°C for 24 hours. Antibody-free medium (DMEM+1% fetal bovine serum) was diluted, added to the cells, and adsorbed at 37°C for 1 hour. The medium containing the virus was discarded, washed 3 times with PBS, and then new DMEM medium containing 1% FBS and 1 mM NAEK was added, and culture was continued at 37°C. On days 1, 2, 3, 4, 5, and 6 after virus infection, the cell supernatant was collected, and the virus titer was determined by plaque assay or qRT-PCR. The results are shown in Figure 3.
  • Embodiment two systematic evaluation 12 kinds of drug-resistant influenza virus strain drug resistance rates
  • the drug resistance rate is evaluated by IC50 value, IC50 is the half-inhibition rate, and the standard curve is S-shaped. It refers to the drug concentration corresponding to when the amount of virus in the drug-dosed group is half that of the control sample. The higher the IC50 inhibition, the worse the sensitivity of the influenza virus to the drug.
  • Different drug resistance mutations take A/WSN/33(H1N1) as an example) correspond to different drugs with decreased sensitivity.
  • the determination method is as follows:
  • Drug dilution Dilute the drug according to the appropriate gradient, and the commonly used dilution gradients are 3 times, 5 times, 10 times, etc.
  • Luciferase detection Mix Cell titer-Glo and PBS at a ratio of 1:1, add to the cell plate (50 ⁇ L/well), let it stand for 10 minutes, and perform microplate multifunctional microplate reader (LB 942, Berthold, Germany) reading.
  • transpeptidase reaction using the enzymatic reaction polypeptide fixed-point labeling technology, by inserting small short peptide sequences into the viral genome, using the corresponding enzymes to specifically recognize short peptide sequences and dyes, without affecting the growth kinetics of the virus
  • fluorescent labeling of different proteins of influenza virus is realized. 5 proteins of influenza virus were labeled by 3 kinds of protein site-specific labeling techniques. The labeled influenza virus can be used to monitor the life activity of the virus after entering the cell.
  • the corresponding peptide sequences were specifically inserted into five different plasmid fragments of NA, HA, M1, M2, and NP, NA-ybbR/M2-ybbR, NP-FlAsH/M1-FlAsH, and HA 5Xgly .
  • the double arsenic-tetracysteine labeling technique is used to realize the specific labeling of two internal proteins (NP and M1) of influenza virus.
  • NP and M1 the Tetra cysteine sequence
  • CCXXCC Tetra cysteine sequence
  • Nucleoprotein NP and matrix protein M1 are internal proteins of influenza virus, and FlAsH-EDT2 dye can penetrate the cell membrane and viral envelope to specifically label NP or M1 protein.
  • the specific recognition of the 5xGly sequence by the Sortase A enzyme is used to realize the specific labeling of the HA protein of the influenza virus.
  • the hemagglutinin HA protein specifically recognizes the sialic acid receptor on the cell surface.
  • the HA protein plays a role in determining the antigenicity of the influenza virus and the process of the virus entering the host cell.
  • the labeling of the HA protein can be used for the antigenic transformation of the influenza virus and the study of host specificity.
  • ybbR sequence (DSLEFIASKLA) by Sfp synthetase is used to realize the specific labeling of influenza virus NA protein and M2 protein.
  • Labeling of NA can be used to study the virus shedding process.
  • NA releases the virus from host cells by cleaving sialic acid on the host cell surface.
  • Marking of M2 can be used to study the genome release process after virus-infected cells.
  • the M2 ion channel mediates the proton acidification process, which fuses the viral envelope with the endosomal membrane and releases the viral genome into the cell.
  • the specific construction method is as follows, based on the influenza virus 12 plasmid reverse genetics system, using the pHH21-NP plasmid as a template, using the Q5 site-directed insertion kit to insert the CCPGCC short peptide sequence; After the recombination method was connected to the stop codon in the NP plasmid containing the CCPGCC sequence obtained in the previous step, the pHH21-NP/FlAsH marker plasmid was obtained. Using pHH21-M1 in the influenza virus 12 plasmid reverse genetics system as a template, using the Q5 site-directed insertion kit, the sequence to be inserted was linked to both ends of the PCR primers for synthesis. Primer sequences are shown in Table 2.
  • the HA protein of influenza virus will be hydrolyzed into two subunits, HA1 and HA2, by TPCK enzyme. Based on this characteristic of HA protein, we inserted 5 Gly sequences before the hydrolysis position of TPCK. The insertion of the short peptide sequence was also implemented using the Q5 site-directed insertion kit.
  • the construction of the plasmid takes pHH21-NA as a template, and the construction of the pHH21-NA/ybbR plasmid is realized by inserting a ybbR segment and a repeat segment (packaging signal) through homologous recombination.
  • the construction of the pHH21-NA/ybbR plasmid takes pHH21-NA as a template, inserts the ybbR fragment and the repeating fragment (packaging signal) through homologous recombination to realize the construction of the pHH21-NA/ybbR plasmid, and the primer sequence constructed by the pHH21-NA/ybbR plasmid is as follows: Table 2 shows.
  • influenza virus M plasmids jointly encode the M1 protein and the M2 protein, the open reading frames of the two overlap. There is a total of 45 nucleotides between M1 and M2, including the C-terminus of M1 and most of the extracellular domain of M2 (residues 10-25).
  • a ybbR tag into the M2 ectodomain after the M1 stop codon. But disulfide bonds (C17 and C19 in M2) and glycosylation (N20 site) upstream of the inserted tag significantly reduced labeling efficiency, possibly due to blocking tag accessibility.
  • C17S, C19S and C20S mutations To improve labeling, we introduced C17S, C19S and C20S mutations.
  • the mutations of C17S and N20S in M1 are nonsense mutations, but C19S leads to the mutation of M248I in M1.
  • Experimental results showed that the mutation did not affect the fitness or morphology of the virus in vitro, while the introduced mutation significantly improved the markers of M2.
  • the primer sequences constructed from the pHH21-M2/ybbR plasmid are shown in Table 2.
  • the plasmid point mutation primer sequences of five different influenza virus segment proteins fluorescently labeled in Table 2 are shown in SEQ ID NO: 13-20 in the sequence listing.
  • Probes for enzyme (Sortase A and Sfp synthase) catalyzed labeling reactions were prepared at room temperature: dyes (Alexa-555maleimide, Alexa-647maleimide, DyLight 405maleimide) were dissolved in anhydrous dimethylformamide (anhydrous DMF) to the final concentration is 15mM. Dissolve CLPETGG short peptide (Gene Script) or CoA in PBS containing 2mM EDTA to a final concentration of 10mM. The two were mixed until the final concentration of CLPETGG short peptide/CoA was 5 mM, and the final concentration of the dye was 7.5 mM.
  • MDCK cells were infected with labeled influenza virus at 33°C.
  • composition of the labeled reaction system is as follows (based on Opti-MEM): 0.25% BSA, 5 mM CaCl 2 , 5 mM MgCl 2 , 200 ⁇ M SrtA, 5 ⁇ M Sfp, 50 ⁇ M CLPETGG probe, 2.5 ⁇ M CoA probe.
  • DRRIV virus can be used to neutralize both non-drug-resistant and drug-resistant strains.
  • Embodiment 5 Systematic testing of the therapeutic effect of DRRIV virus on wild-type virus and drug-resistant virus
  • Cell preparation spread MDCK in a 96-well plate, and the number of cells per well is 5 ⁇ 10 4 .
  • MOI of WT virus 1
  • MOI of DRRIV virus 0, 0.25, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 8 wells in each group .
  • the final volume of WT virus and DRRIV virus is 100 ⁇ L
  • the culture medium is influenza virus maintenance medium (DMEM+1% fetal calf serum+2 ⁇ g/mLTPCK-trypsin).
  • influenza virus maintenance medium DMEM+1% fetal calf serum+2 ⁇ g/mLTPCK-trypsin
  • MDCK cells were plated in a 6-well plate for use, and the number of cells per well was 2.0 ⁇ 10 5 .
  • Virus infection Select two values of IC50 and IC90, mix DRRIV virus and wild-type virus and add it to the cells, and observe the changes in cell morphology. When the WT virus is completely neutralized, it will not cause cell death and produce cytopathic effect (CPE). The specific results are shown in FIG. 7 .
  • Example 6 Based on the influenza virus visualization method, the neutralization effect of different DRRIV virus strains on wild-type drug-resistant influenza virus strains was verified.
  • a. Cell plating MDCK cells were spread in a Confocol culture dish (27mm), and the number of cells per well was 2.0 ⁇ 10 4 .
  • Fluorescent staining HA/M2 double fluorescent staining was performed at 12h, 24h and 36h after virus infection, respectively.
  • Embodiment 7 testing the promoting effect of NA inhibitors (NA Inhibitors, NAIs) on influenza virus multiple infection.
  • NA inhibitors NA Inhibitors, NAIs
  • DRRIV-1 and DRRIV-3 as representatives to verify PAI (baloxavir, BXM), NPI (Nucleozin) and NAIs (Oser He Wei, Oseltamivir; Zanamivir, Zanamivir) three types of drugs on the neutralization effect.
  • NAIs can be used as an adjuvant to improve the neutralization of DRRIV virus, and the introduction of the corresponding drug resistance site in DRRIV virus can well protect DRRIV virus from being cleared by drugs .
  • low concentrations of NAIs will not cause drug resistance; when used to neutralize drug-resistant influenza virus, although NAIs have no inhibitory effect on DRRIV virus and drug-resistant influenza virus, however NAIs can promote neutralization by reconstituting the ability of cells to be infected by DRRIV virus.
  • Example 8 Viral protein labeling system of transpeptidase to study the recombination rules of influenza virus.
  • Administration method nasal drop after pentobarbital (1%/50mg/kg) anesthesia.
  • mice per group 9 mice per group
  • Preparation of reagents and materials Prepare 4% PFA solution, dissolve 4g of paraformaldehyde PFA in 100ml sterile PBS, dissolve at 60°C, add a small amount of NaOH, dissolve and store in 4°C; prepare 30% sucrose solution, Dissolve 30g sucrose in 100ml sterile water at 37°C; prepare sucrose/gelatin embedding solution, dissolve 10g sucrose in 1L sterile PBS and store at 4°C, dissolve 7.5g gelatin in 100ml 10% sucrose solution Dissolve at 37°C, aliquot and store at -20°C.

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Abstract

一种利用流感病毒基因组分节段的特性，结合基因密码子扩展技术，构建的耐药株复制缺陷性流感病毒，建立了新型抗流感病毒治疗方法。根据已知的耐药流感病毒株基因组序列，通过分子克隆技术获得12株耐药流感病毒株，评价其对常用抗流感药物的耐药率。在耐药流感病毒株的基础上引入PTC突变构建了7种耐药株复制缺陷型流感病毒株(DRRIV)；还构建了用于检测流感病毒各节段重组率的可视化标记流感病毒。根据7种DRRIV的包装效率、各节段重组率、以及对野生型流感病毒的治疗作用，筛选出效果最好的NA28TAG/H274Y，结果表明其能够与野生流感病毒重组并将PTC掺入到重组子代病毒中从而抑制病毒增殖，具有广谱的中和活性。联合使用小分子NA抑制剂(NAIs)进一步增强了NA28TAG/H274Y对野生型流感病毒的中和效果。小鼠模型中表明NA28TAG/H274Y与奥司他韦对耐药流感病毒产生了联合增效作用。

Description

一种复制缺陷型耐药流感病毒及其核酸节段重组率检测方法 技术领域
本发明属于生物制药领域，尤其涉及一种通过密码子重编码技术构建的耐药株复制缺陷性流感病毒、其治疗耐药流感的应用、以及对该病毒不同节段进行重组率检测的方法。
背景技术
甲型流感病毒遗传多样性的一个主要机制是共感染病毒之间完整基因片段的重组。重组是指多个流感病毒在共同感染同一细胞时多个单股负链RNA基因片段的交换，而形成新型子代病毒。重组后的新毒株可能在病毒复制、致病机制和宿主范围等上发生了变化。由于流感病毒基因不断重组，使得季节性流感不断变化，对抗流感病毒的难度也就不断增加。
对抗流感病毒的重要策略之一是抗病毒药物治疗。目前临床批准使用的抗流感药物可以分为三类，即M2离子通道阻断剂，NA抑制剂和病毒聚合酶(RdRps)抑制剂。然而，流感病毒可以通过基因突变引起抗原漂移，回避了抗体介导的抗原中和，使药物与病毒失去结合位点，或者结合作用降低，从而导致了耐药株的出现。在季节性A(H1N1)病毒株中，H274Y-NA突变是最常见的奥司他韦耐药性标记。在季节性H3N2流感病毒株中，E119V突变最常见，可导致高水平的奥司他韦耐药性。有的流感病毒会对多种神经氨酸酶抑制剂产生耐药。
以最为普遍的H274Y突变为例阐述耐药性的分子机制，NA蛋白的天然底物唾液酸可以与NA的活性位点结合(PDB:2BAT)，当274位点发生H到Y变异的时候，由于疏水性的作用，它使奥司他韦与NA蛋白的距离增加。由于奥司他韦同NA蛋白的结合是缓慢结合，但同时由于侧链构象扭曲，使它的结合不稳定，因此它会出现缓慢结合又快速分离。所以此时要让NA抑制剂与NA蛋白相互作用，就需要更高浓度的NA抑制剂，即病毒出现了耐药性。因此，病毒不同基因片段的重组频率高，病毒重组频发率高，病毒易产生耐药性，成为本领域亟待解决的技术问题之一。
流感病毒的突变也为传统的流感病毒多价灭活疫苗带来了挑战，导致免疫效果的不确定性。周德敏、张礼和团队采用反向遗传学技术构建了免疫效果全面的非复制型疫苗，对人胚肾(HEK)293T细胞进行了改造，利用慢病毒载体转染一种来自原核生物的正交翻译系统，改造后的293T细胞系用于流感病毒的包装。继而通过对流感病毒基因组进行定点突变人工插入终止密码，产生一种所谓PTC(prematureterminationcodon)病毒，这种病毒仍可以正常增殖并出现CPE，利用这种方法对所有8个基因进行突变分析，观察子代病毒的感染力与稳定性。课题组最终选择了一个PTC-4A病毒研究其安全性、免疫原性以及保护效果，PTC-4A病毒的突变没有涉及HA与NA基因，而涉及到PA、PB1、PB2与NP。这种非复制型病毒可以诱导全面的适应性免疫应答，包括高效价的血凝抑制抗体(HI)、中和抗体(NT)与SIgA，此外病毒特异性CTLs数量高于传统疫苗的10倍，免疫保护效果与现行减毒活疫苗类似，甚至优于后者，特别是对不同血清型流感病毒显示有明显的交叉保护作用(Generation of influenza A viruses as live but replication-incompetent virus vaccines.Science,354(6316),1170–1173.)。上述非复制型流感病毒发挥作用依赖于所述非复制型流感病毒毒株与野生型流感病毒流行株之间抗原谱的交叉，通过引发机体免疫应答起到预防流感病毒感染的目的，然 而对已经发生的感染则效果欠佳。首先，由于非复制型流感病毒激发免疫应答需要较长的时间，对已经感染甚至出现了临床症状的患者没有作用。其次，非复制型流感病毒虽然能够通过内源抗原递呈的方式激发更全面的免疫应答，与灭活疫苗相比在细胞免疫应答方面具有优势，但其提供的抗原谱并没有比灭活疫苗更丰富。另外，由于目前抗流感病毒药物的广泛应用，在抑制流感病毒流行株感染的同时也抑制了疫苗株的作用。
发明内容
针对上述现有技术中存在的缺陷，本发明的目的在于提供一种全新的抗病毒策略，在不引起病毒耐药性的前提下，通过密码子重编码技术，基因突变和分子克隆技术构建耐药株复制缺陷性流感病毒(DRRIV)，利用流感病毒分节段和片段重组的天然特性，对野生型流感病毒和耐药株流感病毒均能良好中和从而达到抑制增殖的效果，并验证NA抑制剂对中和效果具有增强作用，筛选出中和效果最佳的耐药流感病毒株/药物组合NA28TAG/H274Y+奥司他韦，并且首次在小鼠水平上进行了该病毒株+药物组合治疗效果的验证。除此外，首次构建流感可视化方法，检测流感病毒不同节段的重组效率，该方法可用于多重耐药性流感病毒的治疗，也可普适用于其他的分节段病毒治疗与基因重组研究。
本发明提供了一种利用流感病毒基因组分节段的特性，结合基因密码子扩展技术，构建的耐药株复制缺陷性流感病毒，建立了新型抗流感病毒治疗方法。根据已知的耐药流感病毒株基因组序列，通过分子克隆技术获得12株耐药流感病毒株，评价其对常用抗流感药物的耐药率。在耐药流感病毒株的基础上引入PTC突变构建了7种耐药株复制缺陷型流感病毒株(DRRIV)；还构建了用于检测流感病毒各节段重组率的可视化标记流感病毒。根据7种DRRIV的包装效率、各节段重组率、以及对野生型流感病毒的治疗作用，筛选出效果最好的NA28TAG/H274Y，结果表明其能够与野生流感病毒重组并将PTC掺入到重组子代病毒中从而抑制病毒增殖，具有广谱的中和活性。联合使用小分子NA抑制剂(NAIs)进一步增强了NA28TAG/H274Y对野生型流感病毒的中和效果。小鼠模型中表明NA28TAG/H274Y与奥司他韦对耐药流感病毒产生了联合增效作用。
具体而言：
一方面，本发明提供一种复制缺陷型耐药流感病毒(DRRIV)，其特征在于通过反向遗传学操作拯救产生的流感病毒，包括至少一种耐药突变以及至少一种PTC突变；其中，所述耐药突变位于PA、NP、NA和/或M2蛋白，所述PTC突变导致DRRIV在天然宿主细胞中无法增殖和传代；但在转基因细胞中、添加非天然氨基酸的情况下能够在插入的PTC位点通读实现增殖传代。
进一步，本发明所述复制缺陷型耐药流感病毒，其特征在于所述至少一种耐药突变位于选自下组的氨基酸残基位点：PA蛋白第38位、PA蛋白第119位、NP蛋白第289位、NA蛋白第119位、NA蛋白第274位、NA蛋白第292位、NA蛋白第294位、M2蛋白第26位、M2蛋白第27位、M2蛋白第31位。
本发明中复制缺陷型耐药流感病毒各蛋白的氨基酸残基编号依据A/WSN/33(H1N1)株确定。
进一步，本发明所述复制缺陷型耐药流感病毒，其特征在于所述至少一种耐药突变选自由PA蛋白I38T、PA蛋白E119D、NP蛋白Y289H、NA蛋白E119V、NA蛋白H274Y、NA蛋白R292K、NA蛋白N294S、M2蛋白L26F、M2蛋白V27A、M2蛋白S31N组成的组。
进一步，本发明前述任一项复制缺陷型耐药流感病毒，其特征在于所述至少一种耐药突变包括两种耐药突变，优选NA蛋白E119V+NA蛋白R292K、NA蛋白E119V+NA 蛋白N294S、NA蛋白H274Y+NA蛋白N294S、NA蛋白H274Y+NA蛋白R292K、NA蛋白R292K+NA蛋白N294S。
进一步，本发明所述复制缺陷型耐药流感病毒，其特征在于所述至少一种PTC突变是在选自下组的氨基酸残基位点插入终止密码子：PB2蛋白第33位、PB1蛋白第52位、PA蛋白第266位、HA蛋白第57位、NP蛋白第101位、NA蛋白第28位、M2蛋白第37位、NS蛋白第131位。
进一步，本发明所述复制缺陷型耐药流感病毒，其特征在于所述至少一种PTC突变是将所述氨基酸残基位点的原始密码子突变为TAG。
进一步，本发明所述复制缺陷型耐药流感病毒，其特征在于包括耐药突变为NA蛋白H274Y、PTC突变为NA蛋白第28位的密码子突变为TAG。
第二方面，本发明提供一种可视化标记的流感病毒，其特征在于采用3种蛋白定点标记技术对流感病毒的多种结构蛋白进行标记；所述3种蛋白定点标记技术分别利用FIAsH-EDT2对四半胱氨酸序列(CCXXCC)、Sortase A酶对5×Gly序列、Sfp合成酶对ybbR序列(DSLEFIASKLA)的特异性识别进行蛋白质标记。
进一步，本发明所述可视化标记的流感病毒，其特征在于通过反向遗传操作将四半胱氨酸序列(CCXXCC)编码核酸插入NP蛋白编码区和M1蛋白编码区、将5×Gly序列编码核酸插入HA蛋白编码区、将ybbR序列(DSLEFIASKLA)插入NA蛋白编码区和M2蛋白编码区。
第三方面，本发明提供流感病毒重组率的检测方法，其特征在于将未标记流感病毒与前述任一项可视化标记的流感病毒共感染宿主细胞，通过检测重组子代可视化信号比例，分析计算重组率。
进一步，本发明所述流感病毒重组率的检测方法，其特征在于所述未标记流感病毒为权利要求1-7中任一项所述复制缺陷型耐药流感病毒，所述重组率为分别编码NP蛋白、M1蛋白、HA蛋白、NA蛋白和M2蛋白的复制缺陷型耐药流感病毒基因组节段。
第四方面，本发明提供前述任一项复制缺陷型耐药流感病毒的基因组核酸，其至少包括分别编码NP、PB1、PB2、PA、M、NS、HA、NA蛋白的基因节段。
第五方面，本发明提供一组质粒，包括分别含有NP、PB1、PB2、PA、M、NS、HA、NA蛋白表达盒的质粒；所述质粒共转染宿主细胞能够包装产生前述任一项复制缺陷型耐药流感病毒颗粒。
第六方面，本发明提供一种组合物，其包括前述任一项的复制缺陷型耐药流感病毒、前述基因组核酸、或前述一组质粒，以及可选的药学上可接受的载体和/或辅料。
进一步，本发明所述组合物，其特征在于还包括有效量的流感病毒抑制剂。
进一步，本发明前述任一项组合物，其特征在于所述流感病毒抑制剂选自NA抑制剂奥司他韦(Oseltamivir)、扎那米韦(Zanamivir)，PA抑制剂巴洛沙韦(Baloxavir)，NP抑制剂Nucleozin，及其衍生物。
第七方面，本发明提供前述任一项复制缺陷型耐药流感病毒、前述的基因组核酸、前述一组质粒、或前述任一项组合物在制备预防和治疗流感病毒感染的药物中的应用。
进一步，本发明所述的应用，其特征在于所述药物为疫苗；所述流感病毒感染包括H1N1亚型流感病毒感染。
进一步，本发明所述的应用，其特征在于所述流感病毒感染包括耐药流感病毒感染和不耐药流感病毒感染。
第八方面，本发明提供流感病毒共感染诱导剂在制备增强复制缺陷型耐药流感病毒对流感病毒中和活性中的应用，其特征在于所述复制缺陷型耐药流感病毒为前述任一项复制缺陷型耐药流感病毒。
进一步，本发明所述流感病毒抑制剂在制备增强复制缺陷型耐药流感病毒对流感病毒中和活性中的应用，其特征在于所述流感病毒共感染诱导剂选自NA抑制剂奥司他韦(Oseltamivir)、扎那米韦(Zanamivir)，PA抑制剂巴洛沙韦(Baloxavir)，NP抑制剂Nucleozin，及其衍生物。
本发明所采用的的技术方案概括如下：
基于反向遗传学体系，本发明首次成功构建12种不同组合的耐药流感病毒株，本发明系统评价12种耐药病毒株对不同小分子药物抑制剂的耐药率。在此基础上，利用密码子重编码技术和分子克隆技术成功构建了7种不同的DRRIV，利用流感病毒分节段和片段高重组的特点，当DRRIV病毒在与野生型病毒共感染时，使含有PTC的病毒基因组得以通过片段重组进入子代病毒中，进而使野生型病毒失去活性(中和)，且DRRIV病毒对野生型病毒和耐药株病毒均有良好的中和作用。并不同方法系统评价其对耐药病毒的中和效果及广谱性，筛选出NA28TAG/H274Y耐药病毒株。
在此基础上，首次使用流感可视化(转肽酶的病毒蛋白标记)的方法验证了DRRIV对病毒的中和作用和NA抑制剂(NAI)通过增强DRRIV进入细胞的能力从而增强中和作用的机制，除此外，还利用该方法成功进行了流感病毒不同节段的重组率检测。并在动物水平上进一步验证NA28TAG/H274Y+奥司他韦的组合对耐药流感病毒的治疗效果。更为具体地，本发明还提供了：
1)12种耐药流感病毒株的包装效率和耐药率评价方法及结果。
2)7种不同的DRRIV的构建方法。
3)7种不同的DRRIV的包装效率，广谱中和效果评价方法及结果。
4)流感病毒可视化构建方法——转肽酶的蛋白标记系统。
5)不同小分子药物抑制剂对中和效果影响及其机制探究方法。
6)转肽酶的病毒蛋白标记系统研究流感病毒重组规律的方法。
7)小鼠水平上评价耐药流感病毒治疗效果的方法。
附图说明
通过参考附图阅读下文的详细描述，本公开示例性实施方式的上述以及其他目的、特征和优点将变得易于理解。在附图中，以示例性而非限制性的方式示出了本公开的若干实施方式，并且相同或对应的标号表示相同或对应的部分，其中：
对每幅图的结果进行描述、如果是结果图还需对每幅结果图产生的原因或要证明的内容进行具体分析。
图1流感病毒不同节段耐药位点示意图
PA,NP,NA,M2四个节段上有不同耐药位点，且对不同耐药抑制剂敏感。
图2 12种不同耐药流感病毒株感染细胞CPE效应
NP,PA耐药流感病毒可成功包装，NA节段耐药流感病毒一个或者两个耐药位点可成功包装，有明显的CPE效应，三个耐药位点及以上，无法成功包装。
图3 12种不同耐药流感病毒株包装和增殖能力
图3a.12种不同耐药流感病毒株的相对包装效率和病毒滴度
图3b.12种不同耐药流感病毒株的生长曲线
不同耐药株与野生型病毒株相比，滴度，生长曲线没有明显差异，包装效率略降低。且耐药位点越多，包装效率则相对越低。
图4 12种不同耐药流感病毒株耐药率测定
收获耐药株病毒液进行耐药性测试，即测定药物BAV对WSN-PA I38T耐药株的抑制率，BAV对WSN-PA/I38T耐药株的抑制率为8.486μM，与其母本毒株WSN相比增加了约18 倍。收获NP-Y289H耐药株病毒液，测定化合物Nucleozin对WSN-NP/Y289H耐药株的抑制率，Nucleozin对WSN-NP/Y289H耐药株的抑制率为20.08μM，与其母本毒株WSN相比增加了约295倍。对成功包装的NA耐药株进行相应的耐药性测试，其中E119V突变可以导致多重耐药性，会导致该耐药株对Peramivir、Oseltamivir和Zanamivir的敏感性显著下降。而H274Y主要引起Oseltamivir耐药，并对Peramivir敏感性也略微下降。R292K主要引起Peramivir和Oseltamivir耐药；N294S主要引起Oseltamivir耐药。两个耐药位点同时引入可以引起多重耐药性。
图5.不同DRRIV构建，耐药率和生长曲线测定
图5a.DRRIV共转染质粒体系
图5b.携带耐药突变位点和PTC位点的DRRIV在培养基中添加NAEK的转基因细胞上的包装效率和病毒滴度
图5c.携带耐药突变位点和PTC位点的DRRIV的耐药率和生长曲线
与普通的耐药流感株相比，携带耐药突变位点和PTC位点的DRRIV在培养基中添加NAEK的转基因细胞上，耐药率和生长曲线均无明显差异；而培养基中不添加NAEK时DRRIV则无法增殖。
图6. 7种不同DRRIV滴度测定及感染细胞CPE效应
图6a 7种包装效率和病毒滴度较高DRRIV的相对包装效率和病毒滴度
图6b 7种包装效率和病毒滴度较高DRRIV在转基因细胞(NAEK)上的CPE
图6c DR-GRV-NA(N28TAG+H274Y)对多种流感病毒株的中和作用
与野生型耐药株相比，不同DRRIV的滴度没有明显差异，包装效率略有不同。
通过细胞的CPE观察，可以发现DR-GRV-NA(N28TAG+H274Y)除了可以中和WSN毒株以外，还可以中和PR8和CA07毒株，在H1N1亚型内实现广谱治疗作用。同时对其他耐药位点突变的NA耐药株野生型也具有中和作用，说明其在对其他耐药株流感也有中和效果。
图7.不同DRRIV对野生型耐药流感病毒的中和作用效果测定
图7a.携带PTC的DRRIV与野生型耐药流感病毒共感染重组产生带PTC突变的子代
图7b.PTC、PTC-PA-I38T、PTC-NP-Y280H、PTC-NA-H274Y对野生型流感、PA-I38T耐药流感、NP-Y280H耐药流感、NA-H274Y耐药流感的中和活性(MOI为1：1)。
图7c.DRRIV(NA-H274Y+NA-28TAG)对H1N1、耐药H1N1、H3N2、B型流感病毒的中和活性，CPE。
7株DRRIV病毒对WT病毒的中和能力存在明显差异，我们计算了不同株的DRRIV对WT流感病毒的半数抑制率，其横坐标表示为二者的感染复数比例。其中PTC位于NP节段比PTC位于NA节段的DRRIV中和效率低，且中和效率最好的DRRIV为
NA-H274Y+NA-28TAG突变组合。
图8.流感可视化方法验证DRRIV对流感病毒的中和作用
图8a.可视化标记流感病毒标记方案
图8b.可视化标记流感病毒与DRRIV共培养被中和。
标记野生型耐药株的M2和HA节段，分别用绿色和红色荧光表示，不同DRRIV中和标记的野生型耐药株后，荧光强度明显降低，且NA-H274Y+NA-28TAG突变组合荧光强度最低，直观的表明DRRIV对野生型耐药流感病毒有中和作用，并进一步验证了NA-H274Y+NA-28TAG突变组合的中和效果最好。
图9.流感可视化方法验证NAI增强DRRIV对野生型耐药流感病毒的中和作用
图9a-9f结果表明NAIs种类的低浓度的药物可以极大地提高DRRIV病毒的中和能力(约3倍)，而PA抑制剂(BXM)或NP抑制剂(Nucleozin)均无此作用。其中Oseltamivir对DRRIV的促进作用最为明显。采用绿色荧光标记的流感病毒和红色荧光 标记的流感病毒接续感染(间隔2h)细胞时，在第12h进行细胞成像，细胞只能被第一种绿色荧光病毒感染。而当外加NAIs(OSV和Zanamivir)时，可以同时看到绿色和红色荧光病毒，而NPI和PAI均无此作用。因此我们得出结论，NAIs可以有效促进细胞的多重感染能力。
图10流感可视化方法检测流感病毒不同节段的重组率。
HA、NA、M为同类方法，重组率为HA>NA>M。而NS、M、NP为同类标记方法，所以重组率为NS>M>NP，综上，在WSN流感病毒中各节段重组效率为HA>NA>M>NP。
图11.小鼠水平上验证NA28TAG/H274Y+奥司他韦组合对耐药流感病毒的治疗效果
a是小鼠实验方案流程图，
b表示小鼠生存率，其中感染DRV-NA未治疗组在第七天全部死亡，但是在加入DR-GRV-NA组中小鼠生存率大大增加，生存率为89％，而在加入DR-GRV-NA+OSV，OSV浓度为1mg/kg，远低于起效浓度，生存率为百分之百。
c表示小鼠经治疗后体重变化，经DR-GRV-NA治疗组和经DR-GRV-NA+OSV治疗组体重在4-5天开始回升，其中DR-GRV-NA+OSV治疗组回复体重比DR-GRV-NA治疗组快，从动物水平证明了DR-GRV-NA与低浓度的OSV联合使用可以增强DR-GRV-NA的治疗效果。
d表示对小鼠肺组织、脑组织研磨，提取组织RNA，通过RT-qPCR检测病毒载量，经DR-GRV-NA+OSV治疗组，肺部和脑部病毒滴度显著下降，与未治疗组有显著性差异。
e、f表示对肺组织冷冻切片，进行免疫荧光检测病毒NP蛋白，治疗组NP蛋白含量明显减少，同时对肺组织石蜡切片，进行HE染色鉴定结果与上述一致。综上，从组织水平验证验证了DR-GRV-NA能有效治疗DRV-NA。
具体实施方式
为了使本发明的目的、技术方案和优点更加清楚，下面将结合附图对本发明作进一步地详细描述。
实施例一、12种耐药流感病毒株的构建及其包装效率的评价
1.1根据不同流感病毒不同种类耐药的突变位点分析和小分子药物抑制剂类型(如图1所示)，通过基因突变和分子克隆方法构建耐药流感病毒株。
首先进行NP,PA,NA节段质粒耐药位点的点突变，再对NA节段不同的耐药突变位点进行组合，具体不同节段耐药突变位点和点突变引物如表1所示。
表1.流感病毒不同节段耐药突变位点点突变引物
Figure PCTCN2022117666-appb-000001
表1中流感病毒不同节段耐药突变位点点突变引物序列如序列表中SEQ ID NO:1-12所示。
1.2将HEK293T传代至6孔板，细胞密度为2.5×10 5/孔，在37℃细胞培养箱中用含10％FBS的DMEM培养基培养24h约长至50％汇合度；
1.3用转染试剂Megatran2.0进行转染，对应于六孔板的每个孔，流感病毒每种节段质粒(加点突变后的节段质粒)加0.15μg，转染试剂加5.4μL，Opti-MEM加180μL。将对应体积的转染试剂与Opti-MEM、质粒与Opti-MEM分别涡旋10秒充分混匀，室温静置5min；然后将上述两种混合物混合涡旋15s充分混匀，室温静置20min后均匀滴加至单层细胞中。
1.4轻轻摇匀，置于37℃5％CO 2孵箱中培养6h后弃去上清，加入2mL的病毒维持培养基(DMEM+1％胎牛血清+2μg/mL TPCK-trypsin)培养。
1.5转染后，每天观察细胞的病变情况，待90％以上细胞病变后收取细胞上清，0.22μm滤膜过滤分装，置于-80℃保存。不同耐药病毒株感染细胞CPE结果见图2。
1.6测定12种不同耐药病毒株滴度和包装效率，记录每天的滴度值，并绘制生长曲线。将细胞传代至6孔板中，细胞密度为4×10 5/孔，在37℃细胞培养箱中用含10％FBS的DMEM培养基培养24h约长满至90％汇合度。弃去细胞培养基，将病毒用无抗的培养基(DMEM+1％胎牛血清)进行稀释后，加入细胞中，37℃吸附1h后弃上清，加入TRIzol裂解，每孔加200μL TRIzol试剂。将上述细胞中的TRIzol裂解液转入1.5mL离心管，室温(15～30℃)放置5min。在上述1.5mL离心管中，按照每1mL TRIzol加0.2mL氯仿的比例加入氯仿，盖紧离心管盖子，用力震荡15s，室温(15～30℃)静置3min，12000g(2～8℃)离心15min。取上层水相置于新的1.5mL离心管，按照每1mL TRIzol加0.5mL异丙醇的比例加入冰冷的异丙醇，室温(15～30℃)静置10min，12000g(2～8℃)离心10min。弃上清，按照每1mL TRIzol加1mL 75％乙醇的比例进行洗涤，涡旋混合，7500g(2～8℃)离心5min，小心观察管壁上沉淀的RNA，弃上清。让沉淀的RNA在室温下自然干燥(RNA沉淀在70％乙醇中可以长期保存)。用预热的20μL的RNAse-Free water溶解RNA沉淀。用Nano-300紫外分光光度计进行RNA定量。同时进行生长曲线的测定，将细胞传代至6孔板，10 6/孔，于37℃培养24h。无抗的培养基(DMEM+1％胎牛血清)进行稀释后，加入细胞中，37℃吸附1h。弃掉含病毒的培养基，用PBS洗3次，然后加入新的含1％FBS、1mM NAEK的DMEM培养基，于37℃继续培养。于病毒感染后的第1、2、3、4、5、6天，收集细胞上清，用噬斑实验或者qRT-PCR测定病毒滴度，结果见图3。
实施例二、系统评价12种耐药流感病毒株耐药率
耐药率以IC50值大小来评价，IC50即半抑制率，标准曲线为S型。是指加药组病毒量为对照样本一半时所对应的药物浓度，IC50抑制越高，说明流感病毒对药物的敏感性越差。不同耐药突变(以A/WSN/33(H1N1)为例)相对应的敏感度下降的药物不同。测定方法如下：
2.1细胞准备：从培养箱中取出MDCK细胞，镜下观察细胞状态和汇合度。待汇合度约为90％时，消化细胞。将细胞培养基吸出后，加入2mL PBS缓冲液洗一次，加入1mL 0.25％Trypsin-EDTA，放入培养箱中静置20min，期间应时不时晃动细胞，避免局部细胞过干。待细胞完全离壁悬浮后，加入5mL含10％FBS的培养基(无抗生素)终止酶活性，混匀后液体转移至15mL离心管，800g离心3min，弃去液体。随后用适量含10％FBS的DMEM重悬细胞后，计数，铺板。96孔板中每孔细胞数为5×10 4个。
2.2加病毒液及药物：将流感病毒与药物混合，其中流感病毒的感染复数为MOI＝0.01，药物进行梯度稀释，使病毒与药物的终体积为100μL，37℃5％CO 2培养箱培养72h。
药物稀释：将药物按合适梯度进行倍比稀释，常用稀释梯度为3倍、5倍、10倍等。
2.3荧光素酶检测：将Cell titer-Glo与PBS以1:1混合，加入细胞板(50μL/孔)，静置10min后，微孔板式多功能酶标仪(LB 942，Berthold，德国)进行读数。
2.4数据分析：绘制IC50曲线，计算IC50、IC90等值。
2.5IC50验证：根据IC50曲线找出IC50理论值，分别取1/2倍、1倍及2倍的理论值进行药物浓度验证，具体结果见图4。
实施例三、对流感病毒五种不同蛋白的实时可视化标记
通过转肽酶反应，利用酶促反应多肽定点标记技术，通过在病毒基因组中插入小的短肽序列，利用相应的酶对短肽序列和染料的特异性识别，在不影响病毒生长动力学的前提下，实现流感病毒不同蛋白的荧光标记。通过3种蛋白定点标记技术，对流感病毒的5个蛋白进行标记。该标记的流感病毒可以用于监测病毒进入细胞后的生命活动。在NA,HA,M1,M2,NP五种不同质粒片段上特异插入对应的肽段序列，NA-ybbR/M2-ybbR，NP-FlAsH/M1-FlAsH，HA 5Xgly。
采用双砷-四半胱氨酸标记技术，实现流感病毒的两种内部蛋白(NP和M1)蛋白的特异性标记。通过分子生物学手段，在NP和M1基因组成功插入Tetra cysteine序列(CCXXCC)，并实现插入外源序列的流感病毒的成功拯救。核蛋白NP和基质蛋白M1为流感病毒的内部蛋白，FlAsH-EDT2染料可以透过细胞膜和病毒包膜，特异性地标记NP或M1蛋白。
采用Sortase A酶对5xGly序列的特异性识别，实现流感病毒的HA蛋白的特异性标记。在流感病毒通过血凝素HA蛋白特异性识别细胞表面的唾液酸受体实验感染。HA蛋白在决定流感病毒抗原性、病毒进入宿主细胞过程等方面起作用，对HA蛋白的标记可用于流感病毒的抗原转化，宿主特异性研究等。
采用Sfp合成酶对ybbR序列(DSLEFIASKLA)的特异性识别，实现流感病毒NA蛋白和M2蛋白的特异性标记。对NA的标记可用于研究病毒释放过程。NA通过裂解宿主细胞表面唾液酸，来实现病毒从宿主细胞的释放。对M2的标记可以用于研究病毒感染细胞后的基因组释放过程。M2离子通道通过介导质子酸化过程，使病毒包膜与与核内体膜融合，实现病毒基因组释放入细胞中。
3.1 NP-FlAsH/M1-FlAsH构建
其具体构建方法如下，基于流感病毒12质粒反向遗传学系统，以pHH21-NP质粒为模板，采用Q5定点插入试剂盒插入CCPGCC短肽序列；对其C端重复片段进行PCR后，采用同源重组方法连入上一步获得的含有CCPGCC序列的NP质粒中终止密码子后，得到pHH21-NP/FlAsH标记质粒。以流感病毒12质粒反向遗传学系统中pHH21-M1为模板，采用Q5定点插入试剂盒，将需要插入的序列连于PCR引物两端进行合成。引物序列如表2所示。
3.2 HA 5XGly质粒构建
在体内环境下，流感病毒的HA蛋白会被TPCK酶作用水解为HA1和HA2两个亚基。基于HA蛋白的这一特性，我们在TPCK水解位置前插入了5个Gly序列。该短肽序列的插入同样采用Q5定点插入试剂盒实现。
3.3 NA-ybbR/M2-ybbR质粒构建
该质粒的构建以pHH21-NA为模板，通过同源重组插入ybbR片段和重复片段(包装信号)实现pHH21-NA/ybbR质粒的构建。pHH21-NA/ybbR质粒的构建以pHH21-NA为模板，通过同源重组插入ybbR片段和重复片段(包装信号)实现pHH21-NA/ybbR质粒的构建，pHH21-NA/ybbR质粒构建的引物序列如表2所示。
由于流感病毒M质粒共同编码M1蛋白和M2蛋白，二者的开放阅读框存在重叠。 M1和M2之间共有45个核苷酸，包括M1C末端和M2胞外域的大部分(残基10-25)。为了尽量减少对M1C端的干扰，我们在M1终止密码子之后将ybbR标签插入M2胞外域。但是插入的标签上游的二硫键(M2中的C17和C19)和糖基化(N20位点)显著降低了标记效率，其原因可能是由于阻断了标签的可达性。为了改善标记，我们引入了C17S、C19S和C20S突变。在M1中C17S和N20S的突变是无义突变的，但是C19S导致了M1中M248I的突变。实验结果显示，该突变在体外并不影响病毒的适应度或形态，而引入的突变显著改善了M2的标记。pHH21-M2/ybbR质粒构建的引物序列如表2所示。
表2.五种不同流感病毒节段蛋白荧光标记的质粒点突变引物
Figure PCTCN2022117666-appb-000002
表2中五种不同流感病毒节段蛋白荧光标记的质粒点突变引物序列如序列表中SEQ ID NO:13-20所示。
3.4可视化流感病毒成像
用于酶(Sortase A and Sfp synthase)催化标记反应的探针在室温下制备：用无水二甲基甲酰胺(anhydrous DMF)溶解染料(Alexa-555maleimide,Alexa-647maleimide,DyLight 405maleimide)至终浓度为15mM。用含2mM EDTA的PBS溶解CLPETGG短肽(Gene Script)或CoA至终浓度为10mM。将二者混合至CLPETGG短肽/CoA终浓度为5mM，染料终浓度为7.5mM。在开始反应大约24h后，在未反应的染料加入2-巯基乙醇，使其最终浓度为10mM，从而使其猝灭。由此得5mM的储存液，其可直接用于病毒及细胞的标记用可视化病毒感染MDCK细胞后，可直接通过病毒实现荧光标记，具体步骤如下：
1)33℃条件下，MDCK细胞以被标记的流感病毒感染。
2)当标记病毒表面蛋白时，在感染后12h，用Opti-MEM清洗细胞，将培养基更换为Opti-MEM+1μM FlAsH-EDT2,将细胞放回33℃继续培养。
3)30min后，将培养基(回收)更换为含1mM EDT2的培养基(33℃，10min)，其将与未结合或非特异结合的FlAsH-EDT2结合形成黑色复合物，以降低背景干扰。
4)更换培养基并清洗细胞。此时，细胞可用于荧光染料标记。
5)将细胞至于冰上10min(为了降低由游离染料的内吞作用引起的背景干扰)。
6)标记的反应体系组成如下(以Opti-MEM为基础)：0.25％BSA,5mM CaCl 2,5mM MgCl 2,200μM SrtA,5μM Sfp,50μM CLPETGG probe,2.5μM CoA probe。
7)冰上标记1h后，细胞用病毒维持培养基洗涤(室温)，然后立即成像。
实施例四、DRRIV构建及其包装效率的评价
我们将PTC位点和耐药位点整合，从而构建复制缺陷型耐药株病毒(Drug-Resistant and Replication-Incompetent Virus，DRRIV)。DRRIV病毒可同时用于非耐药株病毒和耐药株病毒的中和。根据所选TAG突变位点进行相应点突变，按照之前转染，包装病毒的步骤进行病毒的反向遗传学包装，测定滴度和包装效率，并绘制其生长曲线。具体结果见图5、6，引物序列见表3、4。
表3.流感耐药病毒株不同节段蛋白质粒PTC位点选择
Figure PCTCN2022117666-appb-000003
表4.耐药流感病毒株不同节段蛋白质粒PTC位点点突变引物
Figure PCTCN2022117666-appb-000004
Figure PCTCN2022117666-appb-000005
表4中耐药流感病毒株不同节段蛋白质粒PTC位点点突变引物序列如序列表中SEQ ID NO:21-36所示。
实施例五、系统测试DRRIV病毒对野生型病毒和耐药病毒的治疗作用
我们采用不同的方法验证DRRIV病毒对于野生型/耐药株病毒的中和效果，具体操作如下：
5.1细胞活力检测：采用荧光素酶检测试剂盒(Cell titer-Glo，Promega)检测被野生型病毒(WT)及WT+DRRIV病毒感染后的细胞活力，以反映DRRIV病毒的中和能力，操作如下：
1)细胞准备：将MDCK铺于96孔板中，每孔细胞数为5×10 4个。
2)病毒感染：WT病毒的感染复数MOI＝1，DRRIV病毒的感染复数MOI＝0、0.25、0.5、1、5、10、15、20、25、30、35、40，每组8个孔。WT病毒与DRRIV病毒的终体积为100μL，培养基为流感病毒维持培养基(DMEM+1％胎牛血清+2μg/mLTPCK-trypsin)，提前配置好病毒液后，加入到提前24h铺好的细胞板中，每孔100μL。
3)细胞活力测定：只加WT病毒组细胞病变效应达到90％以上时，采用Cell titer-Glo试剂盒检测细胞活力，并计算DRRIV病毒对WT病毒的半数抑制率(IC50)。
5.2细胞病变效应检测：
1)细胞铺板：将MDCK细胞铺于6孔板中备用，每孔细胞数为2.0×10 5个。
2)病毒感染：选取IC50、IC90两个数值，将DRRIV病毒与野生型病毒混合后加入细胞中，观察细胞形态变化。当WT病毒被完全中和时，不会导致细胞死亡，产生细胞病变效应(CPE)，具体结果见图7。
实施例六、基于流感病毒可视化方法验证不同DRRIV病毒株对野生型耐药流感病毒株中和作用效果。
可视化流感病毒的荧光强度：我们成果构建的可视化野生型流感病毒
可用于反应野生型病毒的表达强度，当野生型病毒被DRRIV中和时，荧光强度会逐渐消失，因此我们可以通过监测荧光值来观察野生型病毒的被清楚程度。操作步骤如下：
a.细胞铺板：将MDCK细胞铺于Confocol培养皿(27mm)中，每孔细胞数为2.0×10 4个。b.荧光染色：在病毒感染后12h、24h和36h分别进行HA/M2双荧光染色。c.数据处理：根据荧光图像，计算灰度值，具体结果见图8。
实施例七、测试NA抑制剂(NA Inhibitors，NAIs)对流感病毒多重感染的促进作用。
在外加低浓度(10nM，此浓度下药物对流感病毒不产生抑制作用)药物的情况下，测量了DRRIV病毒的中和能力，以检测抗流感药物对中和效果的影响，由于DRRIV-1/2和DRRIV-3～7可根据其PTC位点分为两类，因此我们采用DRRIV-1和DRRIV-3为代表验证PAI(巴洛沙韦，BXM)、NPI(Nucleozin)和NAIs(奥司他韦，Oseltamivir；扎那米韦，Zanamivir)三类药物对中和效果的影响。基于可视化流感病毒模型，表明利用低浓度的NAIs可以作为佐剂来提高DRRIV病毒的中和作用，并且DRRIV病毒中的相应耐药位点的引入可以很好地保护DRRIV病毒不被药物作用而清除。当用于中和WT型病毒时，低浓度的NAIs不会引起耐药性；当用于中和耐药株流感病毒时，NAIs对DRRIV病毒和耐药株流感病毒虽然均无抑制作用，然而NAIs可以通过重建细胞被DRRIV病毒的感染能力，来促进其中和能力。
为进一步验证药物对DRRIV中和效果的促进作用，我们采用绿色荧光标记的野生型流感病毒和红色荧光标记的复制缺陷流感病毒接续感染(间隔2h)细胞时，在第12h进行细胞成像，细胞只能被第一种绿色荧光病毒感染。而当外加NAIs(OSV和Zanamivir)时，可以同时看到绿色和红色荧光病毒，而NPI和PAI均无此作用。因此我们得出结论，NAIs可以有效促进细胞的多重感染能力，而DRRIV病毒与WT病毒共同感染同一细胞是发生重组的前提条件，具体结果见图9。
实施例八、转肽酶的病毒蛋白标记系统研究流感病毒重组规律。
将可视化标记流感病毒与未标记流感病毒以1：1，MOI＝1的感染复数感染宿主细胞MDCK。24-48h后收取子代病毒检测病毒滴度。在96孔板中接种MDCK细胞10000个/孔，子代病毒以MOI＝0.15的感染复数，感染MDCK。12小时后，通过计算荧光标记病毒与野生型病毒的重组子代病毒的荧光比例，再通过高内涵分析，定量计算荧光节段重组率，具体结果见图10。
实施例九、小鼠水平上进一步实验验证NA抑制剂-最佳耐药组合的治疗效果
选择4-6周龄BALB/C雌鼠。
给药方式：戊巴比妥(1％/50mg/kg)麻醉后滴鼻。
分组：每组9只小鼠
1)DR-V组(阳性对照)
2)PBS组(阳性对照)
3)DR-V+DR-GRV
4)DR-V+DR-GRV+OSV
5)DR-GRV
检测指标：
1)记录14天内生存率及体重变化(体重低于30％时人道主义处死)。
2)day 3取小鼠脑和肺组织，检测免疫荧光及病毒载量。
冰冻切片免疫荧光
试剂和材料的准备:制备4％PFA溶液，取4g多聚甲醛PFA溶解于100ml无菌PBS中，60℃溶解，可加少量NaOH，溶解后分装保存于4℃；制备30％蔗糖溶液，取30g蔗糖溶解于100ml无菌水中，37℃溶解；制备蔗糖/明胶包埋液，取10g蔗糖溶解于1L无菌PBS中，储存于4℃，取7.5g明胶溶于100ml 10％的蔗糖溶液中，37℃溶解，分装并储存于-20℃。
1)用1ml剪开的枪头小心吸取类脑器官，转移至1.5ml EP管中，用1ml DPBS轻轻清洗3次，加入1ml 4％PFA溶液，室温静置30分钟固定组织，时间可根据组织大小做适当调整。
2)小心弃去PFA溶液，用1ml DPBS清洗3次，每次静置5分钟。
3)清洗结束后，加入1ml 30％蔗糖溶液，4℃脱水过夜，让组织沉降。
4)第二天待组织沉降于管底部，小心吸取类器官转移至包埋模具中，37℃预热蔗糖/明胶溶液20-30分钟，待其溶解后加入模具完全覆盖底部，放于4℃待其凝固后，取类脑器官放于胶上，4℃凝固后，再覆盖一层蔗糖/明胶溶液。
5)将模具放置4℃，20分钟，使蔗糖/明胶溶液完全凝固。
6)取出模具，用手术刀片小心切割带有类器官的区域，这一步可以组织结构，固定后的组织在之后切片染色中效果更佳。
7)准备冰冻浴，泡沫箱中加入液氮，将装有异戊烷的小烧杯放置其中，明胶包裹的类器官再次放入模具中，加OCT包埋剂，用镊子将模具伸入异戊烷中为防止冻裂，可多次上下移动，直至组织完全冷冻。
8)将组织固定在冰冻切片机上，进行切片，在找组织面的过程中使用50μm厚度，当组织出现后改为8-10μm厚度，并将刀片移至未使用过的位置，保证切片完整没有刀痕，切片可储存在-80℃。
9)配制PBST溶液，PBS粉末溶于1L去离子水中，加入终浓度0.2％的Triton，超声混匀。
10)切片放入清洗盒中，加入PBST覆盖表面，摇床50rpm/min透膜30分钟。
11)取出切片擦拭干净，用免疫组化笔圈出组织所在位置，滴加封闭羊血清/驴血清覆盖表面，室温封闭1小时。笔蜡的轮廓会将封闭液保持在组织切片上，注意不要直接滴加在组织上。
12)吸走封闭液，滴加一抗稀释液，4℃湿盒孵育过夜。
13)取出切片，用PBST冲洗三次，单独冲洗避免交叉污染。
14)切片擦拭干净，滴加对应的二抗，转移至湿盒中室温避光孵育1小时，为避免荧光淬灭，后续步骤均需避光操作。
15)取出切片，用PBST冲洗三次，单独冲洗避免交叉污染。
16)切片擦拭干净，滴加抗荧光淬灭液(含DAPI)，盖玻片封片，及时成像，镜下观察前存放在4℃。结果如图11所示。
以上介绍了本发明的较佳实施方式，旨在使得本发明的精神更加清楚和便于理解，并不是为了限制本发明，凡在本发明的精神和原则之内，所做的修改、替换、改进，均应包含在本发明所附的权利要求概括的保护范围之内。

Claims (6)

  1. 一种复制缺陷型耐药流感病毒(DRRIV)，其是通过反向遗传学操作拯救产生的流感病毒，包括耐药突变以及PTC突变；
    其中，所述耐药突变选自NA蛋白H274Y+NA蛋白N294S、NA蛋白H274Y+NA蛋白R292K、NA蛋白E119V+NA蛋白H274Y、NP蛋白Y289H、PA蛋白I38T、NA蛋白H274Y或NA蛋白N294S，所述PTC突变导致DRRIV在天然宿主细胞中无法增殖和传代；
    PTC突变是在NA蛋白第28位突变为终止密码子。
  2. 如权利要求1所述复制缺陷型耐药流感病毒，其特征在于耐药突变为NA蛋白H274Y、PTC突变为NA蛋白第28位的密码子突变为TAG。
  3. 一种可视化标记的流感病毒，其特征在于，所述流感病毒为如权利要求1-2中任一项所述复制缺陷型耐药流感病毒，采用3种蛋白定点标记技术对流感病毒的多种结构蛋白进行标记；所述3种蛋白定点标记技术分别利用FIAsH-EDT2对四半胱氨酸序列(CCXXCC)、Sortase A酶对5×Gly序列、Sfp合成酶对ybbR序列(DSLEFIASKLA)的特异性识别进行蛋白质标记；
    通过反向遗传操作将四半胱氨酸序列(CCXXCC)编码核酸插入NP蛋白编码区和M1蛋白编码区、将5×Gly序列编码核酸插入HA蛋白编码区、将ybbR序列(DSLEFIASKLA)插入NA蛋白编码区和M2蛋白编码区。
  4. 流感病毒重组率的检测方法，其特征在于将未标记流感病毒与权利要求3所述可视化标记的流感病毒共感染宿主细胞，通过检测重组子代可视化信号比例，分析计算流感病毒重组率。
  5. 一种组合物，其包括
    (1)权利要求1-2中任一项所述复制缺陷型耐药流感病毒；
    (2)可选的，药学上可接受的载体和/或辅料；
    (3)可选的，有效量的流感病毒抑制剂；
    其中，所述流感病毒抑制剂选自N A抑制剂奥司他韦(Oseltamivir)、扎那米韦(Zanamivir)，PA抑制剂巴洛沙韦(Baloxavir)，NP抑制剂Nucleozin。
  6. 权利要求1-2中任一项所述复制缺陷型耐药流感病毒、或权利要求5所述组合物在制备预防和治疗流感病毒感染的药物中的应用；
    其中，所述流感病毒感染包括耐药流感病毒感染和不耐药流感病毒感染。
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