CN113755456A - Replication-defective drug-resistant influenza virus and nucleic acid segment recombination rate detection method thereof - Google Patents
Replication-defective drug-resistant influenza virus and nucleic acid segment recombination rate detection method thereof Download PDFInfo
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Abstract
The invention relates to a replication-defective drug-resistant influenza virus and a nucleic acid segment recombination rate detection method thereof. According to the genome sequence of the drug-resistant influenza virus strain, 12 drug-resistant influenza virus strains are obtained by a molecular cloning technology, and the drug resistance rate of the drug-resistant influenza virus strains on anti-influenza drugs is evaluated. The invention constructs 7 drug-resistant strain replication-defective influenza virus strains (DRRIV); and the visual marker influenza virus for detecting the recombination rate of each segment of the influenza virus is also constructed. The most effective NA28TAG/H274Y was selected. The combined use of small molecule NA inhibitors (NAIs) further enhanced the neutralizing effect of NA28TAG/H274Y on wild-type influenza virus. The NA28TAG/H274Y and oseltamivir are shown to produce combined synergistic effect on drug-resistant influenza virus in a mouse model.
Description
Technical Field
The invention belongs to the field of biological pharmacy, and particularly relates to a drug-resistant strain replication-defective influenza virus constructed by a codon recoding technology, application of the drug-resistant strain replication-defective influenza virus in treating drug-resistant influenza, and a method for detecting recombination rates of different segments of the virus.
Background
One of the major mechanisms of influenza a virus genetic diversity is the recombination of complete gene segments between co-infected viruses. Recombination refers to the exchange of multiple single-stranded negative-strand RNA gene segments to form a new progeny virus when multiple influenza viruses co-infect the same cell. The recombinant new strain may have changed in virus replication, pathogenic mechanism, host range, etc. Due to the continuous recombination of influenza virus genes, seasonal influenza changes continuously, and the difficulty of resisting influenza virus is increased continuously.
One of the important strategies against influenza virus is antiviral drug therapy. Anti-influenza drugs currently in clinical approval for use can be divided into three classes, namely M2 ion channel blockers, NA inhibitors and inhibitors of viral polymerase (RdRps). However, influenza viruses can cause antigenic drift through genetic mutation, circumventing antibody-mediated antigen neutralization, causing the drugs to lose binding sites with the virus, or the binding effect to decrease, leading to the emergence of resistant strains. In seasonal A (H1N1) strains, the H274Y-NA mutation is the most common marker of oseltamivir resistance. In seasonal strains of H3N2 influenza, the E119V mutation is most common and can lead to high levels of oseltamivir resistance. Some influenza viruses develop resistance to a variety of neuraminidase inhibitors.
Taking the most common H274Y mutation as an example to illustrate the molecular mechanism of drug resistance, the natural substrate sialic acid of the NA protein can bind to the active site of NA (PDB:2BAT), and when the H to Y variation occurs at position 274, it increases the distance between oseltamivir and the NA protein due to the hydrophobic effect. Since oseltamivir binds slowly to the NA protein, but at the same time its binding is unstable due to side chain conformational distortions, it undergoes slow binding and rapid dissociation. Therefore, in order to allow the NA inhibitor to interact with the NA protein, a higher concentration of the NA inhibitor is required, i.e. the virus is resistant. Therefore, the recombination frequency of different gene segments of the virus is high, the recombination frequency of the virus is high, and the virus is easy to generate drug resistance, which becomes one of the technical problems to be solved in the field.
The mutation of the influenza virus also brings challenges to the traditional polyvalent inactivated influenza virus vaccine, and leads to uncertainty of immune effect. Zhoudmin, Zhang Li and team adopt reverse genetics technology to construct a non-replicative vaccine with comprehensive immune effect, Human Embryonic Kidney (HEK)293T cell is modified, a lentiviral vector is used for transfecting an orthogonal translation system from prokaryotes, and the modified 293T cell line is used for packaging influenza virus. Then, a termination code is artificially inserted by site-directed mutagenesis on the genome of the influenza virus to generate a so-called PTC (Positive temperature coefficient) virus which can still normally proliferate and produce CPE, and mutation analysis is carried out on all 8 genes by using the method to observe the infectivity and stability of the progeny virus. The subject group finally selects a PTC-4A virus to research the safety, immunogenicity and protective effect thereof, and the mutation of the PTC-4A virus does not relate to HA and NA genes but relates to PA, PB1, PB2 and NP. The non-replicating virus can induce comprehensive adaptive immune response, including high titer hemagglutination inhibition antibody (HI), neutralizing antibody (NT) and SIgA, in addition, the number of virus-specific CTLs is 10 times higher than that of the traditional vaccine, the immune protection effect is similar to or even better than that of the current attenuated live vaccine, and particularly, the non-replicating virus shows obvious cross protection effect on different serotypes of influenza viruses (Generation of influenza A viruses as live vaccine-in virus vaccines. science,354(6316), 1170-1173.). The non-replicative influenza virus plays a role by depending on the intersection of antigen spectrums between a non-replicative influenza virus strain and a wild influenza virus epidemic strain, and plays a role in preventing influenza virus infection by inducing immune response of an organism, but has a poor effect on the infection which already occurs. First, because the non-replicating influenza virus takes a long time to elicit an immune response, it has no effect on patients who have already been infected or even have clinical symptoms. Secondly, although non-replicating influenza viruses can stimulate a more comprehensive immune response by means of endogenous antigen presentation, they have advantages over inactivated vaccines in terms of cellular immune responses, but they do not provide an antigen profile that is richer than that of inactivated vaccines. In addition, due to the wide application of the current anti-influenza virus medicines, the effects of vaccine strains are also inhibited while the infection of influenza virus epidemic strains is inhibited.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a brand-new antiviral strategy, construct a drug-resistant strain replication-defective influenza virus (DRRIV) by a codon recoding technology, a gene mutation technology and a molecular cloning technology on the premise of not causing virus resistance, well neutralize both wild influenza virus and the drug-resistant strain influenza virus by utilizing the natural characteristics of segmental and segment recombination of the influenza virus so as to achieve the effect of inhibiting proliferation, verify that an NA inhibitor has an enhancement effect on the neutralization effect, screen out a drug-resistant influenza virus strain/drug combination NA28TAG/H274Y + oseltamivir with the best neutralization effect, and verify the treatment effect of the virus strain + drug combination on the level of a mouse for the first time. Besides, an influenza visualization method is constructed for the first time, the recombination efficiency of different segments of the influenza virus is detected, and the method can be used for treating the multi-drug resistant influenza virus and can also be widely applied to other segmented virus treatment and gene recombination research.
The invention provides a novel anti-influenza virus treatment method which is established by constructing a drug-resistant strain replication-defective influenza virus by utilizing the segmented characteristics of an influenza virus genome and combining a gene codon expansion technology. According to the known genome sequence of the drug-resistant influenza virus strain, 12 drug-resistant influenza virus strains are obtained by a molecular cloning technology, and the drug resistance rate of the drug-resistant influenza virus strains to common anti-influenza drugs is evaluated. Introducing PTC mutation on the basis of drug-resistant influenza virus strains to construct 7 drug-resistant strain replication-defective influenza virus strains (DRRIV); and the visual marker influenza virus for detecting the recombination rate of each segment of the influenza virus is also constructed. According to the packaging efficiency, the recombination rate of each segment and the treatment effect on the wild influenza virus of 7 DRRIV, the NA28TAG/H274Y with the best effect is screened, and the result shows that the recombinant DNA TAG/H274Y can be recombined with the wild influenza virus and the PTC is incorporated into recombinant progeny virus so as to inhibit the virus proliferation, and has broad-spectrum neutralization activity. The combined use of small molecule NA inhibitors (NAIs) further enhanced the neutralizing effect of NA28TAG/H274Y on wild-type influenza virus. The NA28TAG/H274Y and oseltamivir are shown to produce combined synergistic effect on drug-resistant influenza virus in a mouse model.
Specifically, the method comprises the following steps:
in one aspect, the invention provides a replication-deficient drug-resistant influenza virus (DRRIV) characterized by rescue of the influenza virus produced by reverse genetic manipulation, comprising at least one drug-resistant mutation and at least one PTC mutation; wherein the drug resistance mutation is located in PA, NP, NA and/or M2 protein, and the PTC mutation causes DRRIV to be incapable of proliferation and passage in natural host cells; but proliferation passage can be realized by reading through the inserted PTC locus under the condition of adding unnatural amino acids in the transgenic cells.
Further, the replication deficient drug resistant influenza virus according to the present invention is characterized in that the at least one drug resistant mutation is located at an amino acid residue position selected from the group consisting of: the 38 th site of PA protein, the 119 th site of PA protein, the 289 th site of NP protein, the 119 th site of NA protein, the 274 th site of NA protein, the 292 th site of NA protein, the 294 th site of NA protein, the 26 th site of M2 protein, the 27 th site of M2 protein and the 31 th site of M2 protein.
The amino acid residue numbers of the proteins of the replication-defective drug-resistant influenza virus of the present invention are determined according to the A/WSN/33(H1N1) strain.
Further, the replication deficient drug resistant influenza virus according to the present invention is characterized in that the at least one drug resistant mutation is selected from the group consisting of 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, M2 protein S31N.
Further, the replication deficient resistant influenza virus according to any one of the preceding claims of the present invention, characterized in that said at least one resistant mutation comprises two 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.
Further, the replication deficient drug resistant influenza virus according to 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 position selected from the group consisting of: the 33 th site of PB2 protein, the 52 th site of PB1 protein, the 266 th site of PA protein, the 57 th site of HA protein, the 101 th site of NP protein, the 28 th site of NA protein, the 37 th site of M2 protein and the 131 th site of NS protein.
Further, the replication-deficient drug-resistant influenza virus of the present invention is characterized in that the at least one PTC mutation is a mutation of the original codon of the amino acid residue site to TAG.
Furthermore, the replication-defective drug-resistant influenza virus is characterized by comprising a drug-resistant mutation of NA protein H274Y and a PTC mutation of NA protein 28 th codon TAG.
In a second aspect, the invention provides a visually marked influenza virus, which is characterized in that a plurality of structural proteins of the influenza virus are marked by adopting a 3-protein site-specific marking technology; the 3 protein site-directed labeling technology respectively utilizes FIAsH-EDT2 to carry out protein labeling on the specificity recognition of a tetra-cysteine sequence (CCXXCC), a Sortase A enzyme to a 5XGly sequence and a Sfp synthetase to a ybbR sequence (DSLEFIASKLA).
Further, the visually labeled influenza virus according to the present invention is characterized in that a four-cysteine sequence (CCXXCC) encoding nucleic acid is inserted into the NP protein-encoding region and the M1 protein-encoding region, a 5 × Gly sequence-encoding nucleic acid is inserted into the HA protein-encoding region, and a ybbR sequence (DSLEFIASKLA) is inserted into the NA protein-encoding region and the M2 protein-encoding region by reverse genetic manipulation.
In a third aspect, the present invention provides a method for detecting the recombination rate of influenza viruses, which is characterized in that unlabeled influenza viruses and any one of the visually labeled influenza viruses co-infect host cells, and the recombination rate is analyzed and calculated by detecting the ratio of visual signals of recombinant progeny.
Further, the method for detecting the influenza virus recombination rate according to the present invention is characterized in that the unlabeled influenza virus is the replication-deficient drug-resistant influenza virus according to any one of claims 1 to 7, and the recombination rate is a replication-deficient drug-resistant influenza virus genome segment encoding the NP protein, the M1 protein, the HA protein, the NA protein, and the M2 protein, respectively.
In a fourth aspect, the present invention provides a genomic nucleic acid of any one of the aforementioned replication-defective, drug-resistant influenza viruses comprising at least the gene segments encoding the NP, PB1, PB2, PA, M, NS, HA, NA proteins, respectively.
In a fifth aspect, the present invention provides a set of plasmids comprising protein expression cassettes for NP, PB1, PB2, PA, M, NS, HA, NA, respectively; the plasmid co-transfected host cell is capable of packaging to produce any of the aforementioned replication-defective, drug-resistant influenza virus particles.
In a sixth aspect, the present invention provides a composition comprising a replication-defective drug-resistant influenza virus of any one of the preceding claims, a genomic nucleic acid of the preceding claims, or a set of plasmids of the preceding claims, and optionally a pharmaceutically acceptable carrier and/or adjuvant.
Further, the composition of the present invention is characterized by further comprising an effective amount of an influenza virus inhibitor.
Further, the composition of any of the preceding claims, characterized in that the influenza virus inhibitor is selected from the group consisting of the NA inhibitor Oseltamivir (Oseltamivir), Zanamivir (Zanamivir), the PA inhibitor baroxavir (Baloxavir), the NP inhibitor nucelozin, and derivatives thereof.
In a seventh aspect, the present invention provides a replication-deficient drug-resistant influenza virus, a genomic nucleic acid, a set of plasmids, or a composition of any one of the foregoing for use in the preparation of a medicament for the prevention and treatment of influenza virus infection.
Further, the use according to the present invention is characterized in that the medicament is a vaccine; the influenza virus infection comprises an influenza virus infection of subtype H1N 1.
Further, the use according to the present invention, wherein the influenza virus infection comprises a drug-resistant influenza virus infection and a drug-intolerant influenza virus infection.
In an eighth aspect, the present invention provides a use of an influenza virus co-infection inducer for preparing a medicament for enhancing the neutralizing activity of a replication-defective drug-resistant influenza virus against an influenza virus, wherein the replication-defective drug-resistant influenza virus is any one of the replication-defective drug-resistant influenza viruses.
Further, the invention relates to an application of the influenza virus inhibitor in preparation of drugs for enhancing the neutralizing activity of replication-defective drug-resistant influenza viruses on the influenza viruses, which is characterized in that the influenza virus coinfection inducer is selected from NA inhibitors Oseltamivir (Oseltamivir), Zanamivir (Zanamivir), PA inhibitors baroxavir (Baloxavir), NP inhibitors Nucleozin and derivatives thereof.
The technical scheme adopted by the invention is summarized as follows:
based on a reverse genetics system, the invention successfully constructs 12 drug-resistant influenza virus strains with different combinations for the first time, and the system evaluates the drug resistance rates of the 12 drug-resistant virus strains to different small molecule drug inhibitors. On the basis, 7 different DRRIV are successfully constructed by using a codon recoding technology and a molecular cloning technology, and by using the characteristics of segmented and segmented high recombination of the influenza virus, when the DRRIV is infected with the wild virus together, the virus genome containing PTC is recombined into progeny virus through the segments, so that the wild virus loses activity (neutralizes), and the DRRIV has good neutralization effect on the wild virus and the drug-resistant strain virus. And different methods are used for systematically evaluating the neutralization effect and broad spectrum of the NA28TAG/H274Y drug-resistant virus strain on the drug-resistant virus, so that the NA28TAG/H274Y drug-resistant virus strain is screened out.
On the basis, the method for visualizing influenza (virus protein labeling of transpeptidase) is used for the first time to verify the neutralizing effect of DRRIV on the virus and the mechanism of enhancing the neutralizing effect of an NA inhibitor (NAI) by enhancing the capability of the DRRIV to enter cells, and besides, the method is also used for successfully detecting the recombination rate of different segments of the influenza virus. And further validated the therapeutic effect of the NA28TAG/H274Y + oseltamivir combination on resistant influenza viruses at the animal level. More specifically, the present invention also provides:
1) methods and results for evaluating packaging efficiency and drug resistance of 12 drug-resistant influenza virus strains.
2)7 different DRRIV construction methods.
3)7 different DRRIV packaging efficiency, broad-spectrum neutralization effect evaluation methods and results.
4) A visual construction method of influenza virus-a protein labeling system of transpeptidase.
5) The influence of different small-molecule drug inhibitors on the neutralization effect and the mechanism thereof are explored.
6) A method for researching influenza virus recombination rule by using a virus protein labeling system of transpeptidase.
7) A method for evaluating the therapeutic effect of drug-resistant influenza virus on a mouse level.
Drawings
The above and other objects, features and advantages of exemplary embodiments of the present disclosure will become readily apparent from the following detailed description read in conjunction with the accompanying drawings. Several embodiments of the present disclosure are illustrated by way of example, and not by way of limitation, in the figures of the accompanying drawings and in which like reference numerals refer to similar or corresponding parts and in which:
the results of each graph are described, and if the results are graphs, the reasons for generating each result graph or the contents to be proved need to be specifically analyzed.
FIG. 1 schematic representation of different segment drug resistance sites of influenza virus
The four segments of PA, NP, NA and M2 have different drug-resistant sites and are sensitive to different drug-resistant inhibitors.
FIG. 212 CPE Effect of cells infected with different resistant influenza Virus strains
NP, PA resistant influenza virus can be packed successfully, one or two resistant sites of NA segment resistant influenza virus can be packed successfully, obvious CPE effect is achieved, and three resistant sites and more than three resistant sites can not be packed successfully.
FIG. 312 different drug resistant influenza virus strains packaging and propagation capacities
FIG. 3a.12 relative packaging efficiency and viral titer of different drug-resistant influenza virus strains
FIG. 3b.12 growth curves of different drug-resistant influenza virus strains
Compared with wild virus strains, the titer and the growth curve of different drug-resistant strains have no obvious difference, and the packaging efficiency is slightly reduced. And the more resistant sites, the relatively lower the packaging efficiency.
FIG. 412 determination of drug resistance of different drug resistant influenza strains
And (3) harvesting virus liquid of the drug-resistant strain to perform drug resistance test, namely determining the inhibition rate of the drug BAV on the WSN-PA I38T drug-resistant strain, wherein the inhibition rate of the BAV on the WSN-PA/I38T drug-resistant strain is 8.486 mu M, and the WSN is increased by about 18 times compared with the parent strain WSN. NP-Y289H drug-resistant strain virus liquid is obtained, the inhibition rate of the compound Nucleozin on the WSN-NP/Y289H drug-resistant strain is measured, the inhibition rate of the Nucleozin on the WSN-NP/Y289H drug-resistant strain is 20.08 mu M, and the inhibition rate is increased by about 295 times compared with the WSN of the parent strain. Corresponding resistance tests were performed on successfully packaged NA-resistant strains, where the E119V mutation could lead to multiple resistance, resulting in a significant decrease in the susceptibility of the resistant strains to Peramivir, Oseltamivir, and Zanamivir. While H274Y mainly caused Oseltamivir resistance and slightly decreased susceptibility to Peramivir. R292K primarily elicits Peramivir and Oseltamivir resistance; N294S mainly caused Oseltamivir resistance. The simultaneous introduction of two drug resistant sites can cause multidrug resistance.
FIG. 5 construction of different DRRIV, determination of drug resistance and growth Curve
FIG. 5a DRRIV Co-transfection plasmid System
FIG. 5b packaging efficiency and viral titer of DRRIV carrying drug resistant mutation site and PTC site on transgenic cells supplemented with NAEK in culture medium
FIG. 5c shows that the DRRIV carrying the drug-resistant mutation site and PTC site has no significant difference in drug resistance and growth curve on the transgenic cells with NAEK added in the culture medium, compared with the conventional drug-resistant influenza strain; DRRIV was unable to proliferate without the addition of NAEK to the medium.
FIG. 6.7 different DRRIV titre assays and CPE Effect of infected cells
FIG. 6a 7 relative packaging efficiencies and viral titers for DRRIV with higher packaging efficiencies and viral titers
FIG. 6b CPE of DRRIV on transgenic cells (NAEK) with higher packaging efficiency and virus titer
FIG. 6c neutralization of multiple influenza strains by DR-GRV-NA (N28TAG + H274Y) compared to wild type drug resistant strains, there was no significant difference in the titers of different DRRIV, and the packaging efficiencies were slightly different.
Through CPE observation of cells, DR-GRV-NA (N28TAG + H274Y) can neutralize PR8 and CA07 strains besides WSN strains, and realize broad-spectrum treatment effect in H1N1 subtype. Meanwhile, the compound has a neutralization effect on wild type NA drug-resistant strains with mutant drug-resistant sites, which shows that the compound also has a neutralization effect on other drug-resistant strains.
FIG. 7 neutralization effect assay of different DRRIV on wild-type drug-resistant influenza viruses
FIG. 7a. Co-infection of DRRIV carrying PTC with wild-type drug resistant influenza Virus recombines to produce progeny with PTC mutation
FIG. 7b shows the neutralizing activity of PTC, PTC-PA-I38T, PTC-NP-Y280H, PTC-NA-H274Y against wild-type influenza, PA-I38T resistant influenza, NP-Y280H resistant influenza, and NA-H274Y resistant influenza (MOI 1: 1).
FIG. 7c neutralizing activity of DRRIV (NA-H274Y + NA-28TAG) against H1N1, drug-resistant H1N1, H3N2, influenza B virus, CPE.
The neutralizing capacity of the 7 strains of DRRIV on WT virus is obviously different, and the half inhibition rate of DRRIV of different strains on WT influenza virus is calculated, and the abscissa of the inhibition rate is expressed as the ratio of the complex number of infection of the DRRIV to the WT influenza virus. DRRIV, in which PTC is located in the NP segment is less efficient in neutralization than PTC is located in the NA segment, and the DRRIV with the best neutralization efficiency is the NA-H274Y + NA-28TAG mutant combination.
FIG. 8 influenza visualization methods to verify neutralization of influenza viruses by DRRIV
FIG. 8a. visual labelling of influenza Virus labelling protocol
Figure 8b. visual marker influenza virus was neutralized in coculture with DRRIV.
The M2 segment and the HA segment of the marked wild type drug-resistant strain are respectively represented by green fluorescence and red fluorescence, after different DRRIV neutralizes the marked wild type drug-resistant strain, the fluorescence intensity is obviously reduced, and the fluorescence intensity of the NA-H274Y + NA-28TAG mutation combination is lowest, so that the DRRIV is intuitively shown to have a neutralization effect on wild type drug-resistant influenza viruses, and the neutralization effect of the NA-H274Y + NA-28TAG mutation combination is further verified to be the best.
FIG. 9 influenza visualization method to verify neutralization of wild-type drug resistant influenza virus by NAI enhanced DRRIV
The results in FIGS. 9a-9f show that low concentrations of drugs of the NAIs class can greatly increase the neutralizing capacity of DRRIV virus (about 3-fold), whereas neither PA inhibitor (BXM) nor NP inhibitor (Nucleozin) has this effect. Among them, Oseltamivir has the most obvious effect of promoting DRRIV. When the cells were infected successively (2 h apart) with the green fluorescence-labeled influenza virus and the red fluorescence-labeled influenza virus, the cells were imaged at 12h and only infected with the first green fluorescence virus. When NAIs (OSV and Zanamivir) were added, green and red fluorescent viruses were observed simultaneously, but neither NPI nor PAI had this effect. We therefore concluded that NAIs are effective in promoting multiple infectious capacity of cells.
FIG. 10 influenza visualization method detects recombination rates of different segments of influenza virus.
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, and in conclusion, the recombination efficiency of each segment in the WSN influenza virus is HA > NA > M > NP.
FIG. 11 demonstration of the therapeutic Effect of the NA28TAG/H274Y + oseltamivir combination on drug resistant influenza viruses at the mouse level
a is a flow chart of a mouse experimental scheme,
b represents the survival rate of the mice, wherein the untreated group infected with DRV-NA died completely on the seventh day, but the survival rate of the mice was greatly increased to 89% in the group added with DR-GRV-NA, while the concentration of OSV was 1mg/kg, which is far lower than the effective concentration, and the survival rate was one hundred percent in the group added with DR-GRV-NA + OSV.
c indicates that the body weight of the mice changed after the treatment, and the body weight of the DR-GRV-NA treated group and the DR-GRV-NA + OSV treated group began to rise within 4-5 days, wherein the body weight of the DR-GRV-NA + OSV treated group returned to the body weight faster than that of the DR-GRV-NA treated group, and the animal level demonstrates that the treatment effect of DR-GRV-NA can be enhanced by using DR-GRV-NA in combination with OSV at a low concentration.
d shows that lung tissues and brain tissues of mice are ground, tissue RNA is extracted, the viral load is detected by RT-qPCR, and the pulmonary and brain virus titer is remarkably reduced by a DR-GRV-NA + OSV treatment group and is remarkably different from that of an untreated group.
e. f, performing immunofluorescence detection on the frozen lung tissue slices to detect the NP protein of the virus, wherein the NP protein content of the treatment group is obviously reduced, and performing HE staining on the paraffin sections of the lung tissue to identify the result is consistent with the above result. In conclusion, the DR-GRV-NA is verified to be capable of effectively treating DRV-NA from the tissue level.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings.
EXAMPLES one, construction of 12 drug-resistant influenza Virus strains and evaluation of packaging efficiency thereof
1.1 constructing drug-resistant influenza virus strains by gene mutation and molecular cloning methods according to the analysis of drug-resistant mutation sites of different types of different influenza viruses and the types of small molecule drug inhibitors (as shown in figure 1).
Firstly, the point mutation of the drug-resistant sites of NP, PA and NA segment plasmids is carried out, and then different drug-resistant mutation sites of NA segments are combined, wherein the specific drug-resistant mutation sites and point mutation primers of different segments are shown in Table 1.
TABLE 1 influenza virus different segment drug-resistant mutation site point mutation primer
The sequences of the point mutation primers of the drug-resistant mutation sites of different segments of the influenza virus in the table 1 are shown as SEQ ID NO 1-12 in the sequence table.
1.2 passage of HEK293T to 6-well plates at a cell density of 2.5X 105Well, cultured in DMEM medium containing 10% FBS at 37 ℃ for about 24h to about 50% confluency;
1.3 transfection with the transfection reagent Megatran2.0, 0.15. mu.g of each segment plasmid of influenza virus (segment plasmid after point mutation) was added, 5.4. mu.L of transfection reagent, and 180. mu.L of Opti-MEM was added for each well of six-well plate. Respectively and uniformly mixing the transfection reagent with the corresponding volume with the Opti-MEM and the plasmid with the Opti-MEM by vortexing for 10 seconds, and standing for 5min at room temperature; and mixing the two mixtures, vortexing for 15s, fully and uniformly mixing, standing at room temperature for 20min, and uniformly dropwise adding the mixture into the monolayer cells.
1.4 shaking gently, standing at 37 deg.C with 5% CO2After culturing in an incubator for 6h, the supernatant was discarded, and 2mL of virus maintenance medium (DMEM + 1% fetal bovine serum + 2. mu.g/mL TPCK-tryps in) was added for culture.
1.5 after transfection, observing the pathological change condition of cells every day, collecting cell supernatant after more than 90% of cells are pathological changed, filtering with 0.22 μm filter membrane, subpackaging, and storing at-80 deg.C. The results of CPE infection of cells by different resistant virus strains are shown in fig. 2.
1.6 titer and packaging efficiency of 12 different resistant virus strains were determined, daily titer values were recorded and growth curves were plotted. Cells were passaged into 6-well plates at a cell density of 4X 105Well, at 37 ℃ in cell culture box with 10% FBS DMEM medium 24h about full to 90% confluency. Discarding cell culture medium, diluting virus with non-antibody culture medium (DMEM + 1% fetal calf serum), adding into cells, adsorbing at 37 deg.C for 1 hrThe supernatant was discarded and lysed by adding TRIzol, 200. mu.L of TRIzol reagent per well. The TRIzol lysate in the cells is transferred to a 1.5mL centrifuge tube and left at room temperature (15-30 ℃) for 5 min. Chloroform was added to the 1.5mL centrifuge tube in a ratio of 0.2mL chloroform to 1mL TRIzol, the centrifuge tube lid was closed, the tube was shaken vigorously for 15 seconds, allowed to stand at room temperature (15 to 30 ℃) for 3min, and centrifuged at 12000g (2 to 8 ℃) for 15 min. The upper aqueous phase was placed in a new 1.5mL centrifuge tube, ice-cold isopropanol was added to the tube at a rate of 0.5mL isopropanol per 1mL TRIzol, and the mixture was allowed to stand at room temperature (15-30 ℃) for 10min and centrifuged at 12000g (2-8 ℃) for 10 min. The supernatant was discarded, washed with 1mL of 75% ethanol per 1mL of TRIzol, vortexed, centrifuged at 7500g (2-8 ℃) for 5min, RNA precipitated on the tube wall was carefully observed, and the supernatant was discarded. The precipitated RNA was allowed to dry naturally at room temperature (the RNA precipitate could be stored for a long period in 70% ethanol). The RNA pellet was dissolved with 20. mu.L of RNAse-Free water in a pre-heat. RNA quantification was performed using a Nano-300 UV spectrophotometer. Growth curve measurements were performed simultaneously, and cells were passaged to 6-well plates, 106Perwell, incubated at 37 ℃ for 24 h. After dilution in a medium without antibody (DMEM + 1% fetal calf serum), the cells were added and adsorbed at 37 ℃ for 1 hour. The virus-containing medium was discarded, washed 3 times with PBS, and then fresh DMEM medium containing 1% FBS and 1mM NAEK was added to continue the culture at 37 ℃. At days 1, 2, 3, 4, 5, 6 after virus infection, cell supernatants were collected and virus titers were determined using plaque assay or qRT-PCR, and the results are shown in fig. 3.
Example two, systematic evaluation of 12 resistant influenza virus strains drug resistance rate
The drug resistance was evaluated as the value of IC50, IC50 is the half inhibition rate, and the standard curve is S-shaped. The higher the inhibition of IC50, the lower the sensitivity of influenza virus to drugs, which is the corresponding drug concentration when the virus amount in the drug group is half of the control sample. The drugs with reduced sensitivity corresponding to different drug resistance mutations (for example, A/WSN/33(H1N 1)) are different.
The measurement method is as follows:
2.1 cell preparation: the MDCK cells were taken out from the incubator, and the cell state and the degree of confluence were observed under a microscope. The cells were digested to a confluence of about 90%. After the cell culture medium is aspirated, addWashing once in 2mL PBS buffer, adding 1mL 0.25% Trypsin-EDTA, placing in an incubator, standing for 20min, and shaking the cells at certain time to avoid over-drying of local cells. And after the cells are completely suspended from the wall, adding 5mL of a culture medium (without antibiotics) containing 10% FBS to terminate the enzyme activity, uniformly mixing, transferring the liquid to a 15mL centrifuge tube, centrifuging for 3min at 800g, and discarding the liquid. The cells were then resuspended in the appropriate amount of DMEM containing 10% FBS, counted and plated. The number of cells per well in the 96-well plate was 5X 104And (4) respectively.
2.2 virus liquid and medicine are added: mixing influenza virus with drug, wherein the influenza virus has MOI of 0.01, and diluting with gradient to obtain final volume of 100 μ L of the virus and drug, and 5% CO at 37 deg.C2The incubator is used for 72 h.
Drug dilution: the medicine is diluted in a proper gradient in a multiple ratio, and the common dilution gradient is 3 times, 5 times, 10 times and the like.
2.3 luciferase assay: cell titer-Glo was mixed with PBS at 1:1, added to a Cell plate (50. mu.L/well), allowed to stand for 10min, and read on a microplate reader (LB 942, Berthold, Germany).
2.4 data analysis: the curve of IC50 was plotted, and the equivalent of IC50 and IC90 was calculated.
2.5IC50 verification: finding out theoretical value of IC50 according to IC50 curve, taking 1/2 times, 1 time and 2 times of theoretical value to carry out drug concentration verification, and specific results are shown in figure 4.
Example three real-time visual labelling of five different proteins of influenza Virus
By transpeptidase reaction, utilizing an enzymatic reaction polypeptide fixed-point labeling technology, inserting a small short peptide sequence into a virus genome, and utilizing the specificity recognition of corresponding enzyme on the short peptide sequence and dye, the fluorescent labeling of different proteins of the influenza virus is realized on the premise of not influencing the growth kinetics of the virus. 5 proteins of the influenza virus are marked by a 3-protein fixed-point marking technology. The labeled influenza virus can be used to monitor vital activity of the virus after it enters a cell. The corresponding peptide fragment sequences, NA-ybbR/M2-ybbR, NP-FlAsH/M1-FlAsH and HA, are specifically inserted into five different plasmid fragments of NA, HA, M1, M2 and NP5Xgly。
Specific labeling of two internal proteins (NP and M1) of influenza virus was achieved using a double arsenic-tetra cysteine labeling technique. By means of molecular biology, Tetra cysteine sequence (CCXXCC) is successfully inserted into NP and M1 genomes, and the successful rescue of influenza virus inserted with exogenous sequence is realized. The nucleoprotein NP and the matrix protein M1 are internal proteins of influenza virus, and the FlaSH-EDT2 dye can permeate cell membranes and virus envelopes to specifically mark NP or M1 proteins.
And (3) specifically identifying the 5xGly sequence by using Sortase A enzyme to realize the specific marking of HA protein of the influenza virus. Infection was tested in influenza virus by specifically recognizing cell surface sialic acid receptor via hemagglutinin HA protein. The HA protein plays a role in determining the antigenicity of the influenza virus, the process of the virus entering host cells and the like, and the marker of the HA protein can be used for the antigen transformation of the influenza virus, the host specificity research and the like.
Specific identification of the YbbR sequence (DSLEFIASKLA) by adopting Sfp synthetase realizes specific labeling of influenza virus NA protein and M2 protein. The labeling of NA can be used to study the viral release process. NA achieves release of the virus from the host cell by cleaving sialic acid on the surface of the host cell. The marker for M2 can be used to study the process of genome release after viral infection of cells. The M2 ion channel fuses the virus envelope and the nuclear endosome membrane by mediating the process of protonation, thus realizing the release of the virus genome into cells.
3.1 NP-FlAsH/M1-FlAsH construction
The specific construction method is that based on the influenza virus 12 plasmid reverse genetics system, pHH21-NP plasmid is used as a template, and a Q5 fixed-point insertion kit is adopted to insert CCPGCC short peptide sequence; after PCR is carried out on the C-terminal repetitive fragment, a homologous recombination method is adopted to connect a stop codon in the NP plasmid containing the CCPCC sequence obtained in the last step, and pHH21-NP/FlAsH labeled plasmid is obtained. A Q5 fixed-point insertion kit is adopted by taking pHH21-M1 in an influenza virus 12 plasmid reverse genetics system as a template, and sequences to be inserted are connected to the two ends of a PCR primer for synthesis. The primer sequences are shown in Table 2.
3.1 HA5XGlyPlasmid construction
Under the in vivo environment, HA protein of influenza virus is hydrolyzed into two subunits of HA1 and HA2 by TPCK enzyme. Based on this property of HA protein, we inserted 5 Gly sequences before the TPCK hydrolysis position. The insertion of the short peptide sequence is also realized by using a Q5 site-directed insertion kit.
3.3 NA-ybbR/M2-ybbR plasmid construction
The construction of the plasmid takes pHH21-NA as a template, and inserts a ybbR fragment and a repetitive fragment (packaging signal) through homologous recombination to realize the construction of pHH21-NA/ybbR plasmid. pHH21-NA/ybbR plasmid construction is realized by inserting a ybbR fragment and a repetitive fragment (packaging signal) through homologous recombination by taking pHH21-NA as a template, and the primer sequences constructed by pHH21-NA/ybbR plasmid are shown in Table 2.
Since influenza M plasmids jointly encode the M1 protein and the M2 protein, there is an overlap in the open reading frames of the two. There are 45 nucleotides in total between M1 and M2, including the C-terminus of M1 and the majority of the M2 extracellular domain (residues 10-25). To minimize interference at the C-terminus of M1, we inserted 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, probably due to blocking the accessibility of the tag. To improve labeling, we introduced C17S, C19S, and C20S mutations. Mutations of C17S and N20S in M1 were nonsense mutations, but C19S resulted in a mutation of M248I in M1. The experimental results show that the mutation does not affect the fitness or the morphology of the virus in vitro, and the introduced mutation obviously improves the labeling of M2. The primer sequences constructed from pHH21-M2/ybbR plasmid are shown in Table 2.
TABLE 2 fluorescent-labeled plasmid point mutation primers for five different influenza virus segment proteins
The sequences of the five different influenza virus segment protein fluorescence labeled plasmid point mutation primers in the table 2 are shown as SEQ ID NO 13-20 in the sequence table.
3.4 visual influenza Virus imaging
The probes for the enzyme-catalyzed labeling reactions (Sortase a and Sfp synthsase) were prepared at room temperature: the dye (Alexa-555maleimide, Alexa-647maleimide, Dylight 405maleimide) was dissolved with anhydrous dimethylformamide (anhydrous DMF) to a final concentration of 15 mM. CLPETGG short peptide (Gene Script) or CoA was dissolved in PBS containing 2mM EDTA to a final concentration of 10 mM. Both were mixed to a final concentration of 5mM for CLPETGG oligopeptide/CoA and 7.5mM for the dye. After about 24h from the start of the reaction, 2-mercaptoethanol was added to the unreacted dye to a final concentration of 10mM, thereby quenching it. The 5mM storage solution can be directly used for labeling viruses and cells, can realize fluorescent labeling directly through the viruses after the MDCK cells are infected by the visual viruses, and comprises the following specific steps:
1) MDCK cells were infected with labeled influenza virus at 33 ℃.
2) When the virus surface proteins were labeled, at 12h post-infection, the cells were washed with Opti-MEM, the medium was changed to Opti-MEM + 1. mu.M FlasH-EDT2, and the cells were returned to 33 ℃ for further culture.
3) After 30min, the medium (recovered) was changed to medium containing 1mM EDT2 (33 ℃, 10min), which would bind to unbound or non-specifically bound FlAsH-EDT2 to form a black complex to reduce background interference.
4) The medium was changed and the cells were washed. At this point, the cells are available for fluorescent dye labeling.
5) Cells were placed on ice for 10min (to reduce background interference caused by endocytosis of free dye).
6) The labeled reaction system consisted of (based on Opti-MEM): 0.25% BSA,5mM CaCl2,5mM MgCl2,200μM SrtA,5μM Sfp,50μM CLPETGG probe,2.5μM CoA probe。
7) After 1h on ice, cells were washed with virus maintenance medium (room temperature) and imaged immediately. Example four DRRIV construction and evaluation of packaging efficiency thereof
We integrated the PTC site and the Drug Resistant site to construct a Replication-deficient Drug Resistant strain Virus (DRRIV). DRRIV virus can be used for neutralizing non-drug-resistant strain virus and drug-resistant strain virus at the same time. Corresponding point mutation is carried out according to the selected TAG mutation site, reverse genetics packaging of the virus is carried out according to the steps of transfection and virus packaging, titer and packaging efficiency are determined, and a growth curve is drawn. The results are shown in FIGS. 5 and 6, and the primer sequences are shown in tables 3 and 4.
TABLE 3 selection of PTC sites for different segment protein plasmids of influenza resistant virus strains
TABLE 4 PTC site point mutation primers for different segment protein plasmids of drug-resistant influenza virus strains
The sequence of the PTC locus point mutation primer of different segment protein plasmids of the drug-resistant influenza virus strain in the table 4 is shown as SEQ ID NO 21-36 in the sequence table.
EXAMPLE V systematic testing of DRRIV Virus for therapeutic Effect against wild-type Virus and drug resistant Virus
We adopt different methods to verify the neutralizing effect of DRRIV virus on wild type/drug-resistant strain virus, and the specific operations are as follows:
5.1 cell viability assay: luciferase assay kit (Cell titer-Glo, Promega) was used to measure Cell viability following infection by wild-type virus (WT) and WT + DRRIV virus, to reflect the neutralizing capacity of DRRIV virus, as follows:
1) cell preparation: MDCK was plated in 96-well plates at 5X 10 cells per well4And (4) respectively.
2) Viral infection: the MOI of WT virus was 1, and the MOI of DRRIV virus was 0, 0.25, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, with 8 wells per group. The final volume of WT virus and DRRIV virus is 100 μ L, the culture medium is influenza virus maintenance medium (DMEM + 1% fetal bovine serum +2 μ g/ml LTPCK-tryps in), and after the virus solution is prepared in advance, the virus solution is added into a cell plate paved 24h in advance, and each well is 100 μ L.
3) Cell viability assay: when the cytopathic effect of the WT virus group reaches more than 90%, Cell titer-Glo kit is adopted to detect the Cell viability, and the half inhibition rate of DRRIV on the WT virus is calculated (IC 50).
5.2 cytopathic effect detection:
1) cell plating: MDCK cells were plated in 6-well plates at 2.0X 10 cells/well for future use5And (4) respectively.
2) Viral infection: selecting two values of IC50 and IC90, mixing DRRIV virus and wild type virus, adding into cells, and observing cell morphology change. When the WT virus was completely neutralized, it did not lead to cell death, resulting in cytopathic effect (CPE), as shown in FIG. 7.
Example six, the effect of neutralization of different DRRIV virus strains on wild-type resistant influenza virus strains was verified based on influenza virus visualization methods.
Visualization of fluorescence intensity of influenza virus: the visualized wild-type influenza virus constructed by our efforts can be used for reflecting the expression intensity of the wild-type virus, and the fluorescence intensity gradually disappears when the wild-type virus is neutralized by DRRIV, so we can observe the clarity degree of the wild-type virus by monitoring the fluorescence value. The operation steps are as follows:
a. cell plating: MDCK cells were plated in Confocol dishes (27mm) at 2.0X 10 cells/well4And (4) respectively. b. Fluorescent staining: HA/M2 dual fluorescent staining was performed 12h, 24h and 36h post viral infection, respectively. c. Data processing: from the fluorescence image, the gray value was calculated, and the specific result is shown in fig. 8.
EXAMPLE seven, the enhancement of influenza virus multiple infection by NA Inhibitors (NAIs) was tested.
Under the condition of adding low-concentration (10nM, the concentration of the drug does not generate inhibition effect on influenza virus) drugs, the neutralizing capacity of DRRIV virus is measured to detect the influence of the neutralizing effect of the anti-influenza drugs, and DRRIV-1/2 and DRRIV-3-7 can be divided into two types according to PTC sites, so DRRIV-1 and DRRIV-3 are taken as representatives to verify the influence of three types of drugs including PAI (Barosavir, BXM), NPI (Nucleozin) and NAIs (Oseltamivir, Oseltamivir; zanavir, Zanamivir). Based on a visual influenza virus model, it is shown that low-concentration NAIs can be used as an adjuvant to improve the neutralization effect of DRRIV, and the introduction of corresponding drug-resistant sites in the DRRIV can well protect the DRRIV from being cleared by the action of drugs. When used to neutralize WT-type viruses, low concentrations of NAIs do not cause drug resistance; when used for neutralizing drug-resistant strain influenza virus, the NAIs have no inhibiting effect on DRRIV virus and drug-resistant strain influenza virus, but the NAIs can promote the neutralizing capability of cells by reconstructing the infecting capability of the cells by the DRRIV virus.
To further verify the promotion effect of the drug on DRRIV neutralization effect, when cells are infected successively (interval 2h) by using green fluorescence labeled wild type influenza virus and red fluorescence labeled replication-defective influenza virus, cell imaging is carried out at 12h, and the cells can only be infected by the first green fluorescence virus. When NAIs (OSV and Zanamivir) were added, green and red fluorescent viruses were observed simultaneously, but neither NPI nor PAI had this effect. We therefore concluded that NAIs are effective in promoting multiple infectious capacity of cells, whereas the co-infection of the same cells with DRRIV virus and WT virus is a prerequisite for recombination, and the specific results are shown in fig. 9.
Example eight viral protein marker system of transpeptidase investigated the influenza virus recombination law.
Visually labeled influenza virus was compared to unlabeled influenza virus at a ratio of 1:1, MOI 1. multiplicity of infection infects host cells MDCK. And collecting the progeny virus 24-48h later to detect the virus titer. In 96-well plates, 10000 MDCK cells per well were inoculated, and progeny viruses infected with MDCK at MOI of 0.15. After 12 hours, the fluorescence ratio of the fluorescence labeled virus and the recombinant progeny virus of the wild type virus is calculated, and then the fluorescence segment recombination rate is quantitatively calculated through high content analysis, and specific results are shown in figure 10.
EXAMPLE nine further experiments at the mouse level demonstrate the therapeutic efficacy of the NA inhibitor-optimal drug resistance combination
Female BALB/C mice of 4-6 weeks of age were selected.
The administration mode comprises the following steps: pentobarbital (1%/50 mg/kg) was used as an anesthetic and then dropped into the nose.
Grouping: 9 mice per group
1) DR-V group (Positive control)
2) PBS group (Positive control)
3)DR-V+DR-GRV
4)DR-V+DR-GRV+OSV
5)DR-GRV
Detection indexes are as follows:
1) survival and weight changes were recorded for 14 days (humanitarian sacrifice below 30% body weight).
2) day 3 mouse brain and lung tissues were taken and immunofluorescence and viral load were measured.
Frozen section immunofluorescence
Preparing 4% PFA solution, dissolving 4g of paraformaldehyde PFA in 100ml of sterile PBS, dissolving at 60 ℃, adding a small amount of NaOH, dissolving, packaging and storing at 4 ℃; preparing 30% sucrose solution, dissolving 30g sucrose in 100ml sterile water, and dissolving at 37 deg.C; preparing sucrose/gelatin embedding solution, dissolving 10g sucrose in 1L sterile PBS, storing at 4 deg.C, dissolving 7.5g gelatin in 100ml 10% sucrose solution, dissolving at 37 deg.C, subpackaging and storing at-20 deg.C.
1) Carefully sucking the brain-like organ with a 1ml cut tip, transferring to a 1.5ml EP tube, gently washing with 1ml DPBS for 3 times, adding 1ml of 4% PFA solution, standing at room temperature for 30 minutes to fix the tissue, and the time can be properly adjusted according to the size of the tissue.
2) The PFA solution was carefully discarded and washed 3 times with 1ml DPBS for 5 minutes each.
3) After washing, 1ml of 30% sucrose solution was added, and the tissue was allowed to settle overnight at 4 ℃.
4) The next day, when the tissue is settled at the bottom of the tube, the organoids are carefully sucked and transferred to an embedding mould, the sucrose/gelatin solution is preheated for 20-30 minutes at 37 ℃, the organoids are added into the mould to completely cover the bottom after the organoids are dissolved, the organoids are placed on the glue after the organoids are placed at 4 ℃ for solidification, and a layer of the sucrose/gelatin solution is covered after the organoids are solidified at 4 ℃.
5) The mold was left at 4 ℃ for 20 minutes to completely solidify the sucrose/gelatin solution.
6) The removal of the mold and careful cutting of the organoid bearing area with the surgical blade allows for tissue organization and better staining of the fixed tissue in later sections.
7) Preparing a freezing bath, adding liquid nitrogen into a foam box, placing a small beaker filled with isopentane into the foam box, placing the organoid wrapped by gelatin into a mould again, adding an OCT embedding agent, extending the mould into the isopentane by using forceps to prevent frost cracking, and moving up and down for multiple times until the tissue is completely frozen.
8) Fixing the tissue on a freezing microtome, slicing, using 50 μm thickness in the process of finding the tissue surface, changing to 8-10 μm thickness when the tissue appears, moving the blade to an unused position to ensure that the section is complete without knife mark, and storing the section at-80 ℃.
9) Preparing PBST solution, dissolving PBS powder in 1L deionized water, adding Triton with the final concentration of 0.2%, and ultrasonically mixing.
10) The sections were placed in a washing box, PBST was added to cover the surface, and the membrane was permeabilized for 30 minutes at 50rpm/min with a shaker.
11) Taking out the section, wiping the section, using an immunohistochemical pen to circle the position of the tissue, dropwise adding the closed goat serum/donkey serum to cover the surface, and sealing for 1 hour at room temperature. The contour of the pen wax will keep the sealing fluid on the tissue section, taking care not to drip directly on the tissue.
12) The blocking solution was aspirated off, primary anti-diluent was added dropwise, and incubated overnight in a wet box at 4 ℃.
13) Sections were removed and washed three times with PBST, washed separately to avoid cross contamination.
14) And (3) wiping the section, dropwise adding corresponding secondary antibody, transferring the section into a wet box, incubating for 1 hour at room temperature in a dark place, and performing dark operation in subsequent steps to avoid fluorescence quenching.
15) Sections were removed and washed three times with PBST, washed separately to avoid cross contamination.
16) The section is wiped clean, anti-fluorescence quenching liquid (containing DAPI) is dripped, the section is covered with a glass slide and imaged in time, and the section is stored at 4 ℃ before being observed under a mirror. The results are shown in FIG. 11.
The foregoing describes preferred embodiments of the present invention, and is intended to provide a clear and concise description of the spirit and scope of the invention, and not to limit the same, but to include all modifications, substitutions, and alterations falling within the spirit and scope of the invention as defined by the appended claims.
Sequence listing
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Claims (10)
1. A replication-deficient, drug-resistant influenza virus (DRRIV), which is an influenza virus produced by rescue by reverse genetic manipulation, comprising at least one drug-resistant mutation and at least one PTC mutation;
wherein the drug resistance mutation is located in PA, NP, NA and/or M2 protein, and the PTC mutation causes DRRIV to be incapable of proliferation and passage in natural host cells;
and, the at least one drug resistant mutation comprises 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.
2. The replication deficient resistant influenza virus according to claim 1, wherein said at least one PTC mutation is an insertion of a stop codon at an amino acid residue position selected from the group consisting of: the 33 th site of PB2 protein, the 52 th site of PB1 protein, the 266 th site of PA protein, the 57 th site of HA protein, the 101 th site of NP protein, the 28 th site of NA protein, the 37 th site of M2 protein and the 131 th site of NS protein.
3. The replication-deficient drug-resistant influenza virus of claim 1, comprising a drug-resistant mutation to NA protein H274Y and a PTC mutation to NA protein 28 th codon mutation to TAG.
4. A visual marked influenza virus is characterized in that 3 protein fixed-point marking technologies are adopted to mark a plurality of structural proteins of the influenza virus; the 3 protein fixed-point labeling technology respectively utilizes FIAsH-EDT2 to perform protein labeling on the specificity recognition of a tetra-cysteine sequence (CCXXCC), a Sortase A enzyme to a 5XGly sequence and a Sfp synthetase to a ybbR sequence (DSLEFIASKLA);
the four cysteine sequence (CCXXCC) encoding nucleic acid was inserted into the NP protein coding region and the M1 protein coding region, the 5x Gly sequence encoding nucleic acid was inserted into the HA protein coding region, and the ybbR sequence (DSLEFIASKLA) was inserted into the NA protein coding region and the M2 protein coding region by reverse genetic manipulation.
5. A method for detecting the recombination rate of influenza viruses, which is characterized in that unmarked influenza viruses and the visualized marked influenza viruses of claim 4 are co-infected with host cells, and the recombination rate of the influenza viruses is analyzed and calculated by detecting the visualized signal ratio of the recombination filial generation.
6. The replication deficient resistant influenza virus genomic nucleic acid according to any one of claims 1 to 3 comprising at least the gene segments encoding the NP, PB1, PB2, PA, M, NS, HA, NA proteins, respectively.
7. A group of plasmids comprising plasmids containing NP, PB1, PB2, PA, M, NS, HA and NA protein expression cassettes respectively; co-transfecting the plasmid into a host cell capable of being packaged to produce the replication-defective drug-resistant influenza virus particle of any one of claims 1 to 3.
8. A composition comprising
(1) The replication-deficient, drug-resistant influenza virus of any one of claims 1 to 3, the genomic nucleic acid of claim 6, or the set of plasmids of claim 7;
(2) optionally, a pharmaceutically acceptable carrier and/or adjuvant;
(3) optionally, an effective amount of an influenza virus inhibitor;
wherein the influenza virus inhibitor is preferably selected from the group consisting of NA inhibitor Oseltamivir (Oseltamivir), Zanamivir (Zanamivir), PA inhibitor baroxavir (Baloxavir), NP inhibitor nucelozin, and derivatives thereof.
9. Use of the replication-deficient, drug-resistant influenza virus of any one of claims 1 to 3, the genomic nucleic acid of claim 6, the set of plasmids of claim 7, or the composition of claim 8 for the preparation of a medicament for the prophylaxis and treatment of influenza virus infection;
wherein, the influenza virus infection comprises drug-resistant influenza virus infection and drug-intolerant influenza virus infection, and preferably H1N1 subtype influenza virus infection.
10. The application of influenza virus co-infection inducer in preparing the medicine for strengthening the neutralizing activity of replication-defective drug-resistant influenza virus to influenza virus is characterized in that
The replication-deficient drug-resistant influenza virus according to any one of claims 1 to 3, wherein the influenza virus coinfection inducer is selected from the group consisting of NA inhibitor Oseltamivir (Oseltamivir), Zanamivir (Zanamivir), PA inhibitor baroxavir (Baloxavir), NP inhibitor Nucleozin, and derivatives thereof.
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| CN114464246A (en) * | 2022-01-19 | 2022-05-10 | 华中科技大学同济医学院附属协和医院 | Method for detecting mutation related to genetic increase based on CovMutt framework |
| WO2023036209A1 (en) * | 2021-09-08 | 2023-03-16 | 北京大学 | Replication-defective drug-resistant influenza virus and method for detecting recombination rate of nucleic acid segment thereof |
| WO2023138651A1 (en) * | 2022-01-19 | 2023-07-27 | Versitech Limited | Rationally designed single-round infectious virus and methods of use thereof |
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| CN113755456B (en) | 2022-02-15 |
| WO2023036209A1 (en) | 2023-03-16 |
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