Broad-spectrum anti-influenza a virus siRNA molecules, derivatives thereof, and uses thereof

Abstract

This invention discloses a broad-spectrum anti-influenza A virus siRNA molecule, its derivatives, and their applications, belonging to the field of biotechnology. The siRNA is named siPB2-12 and / or siNP-4, and / or a derivative of the RNA molecule; one strand sequence of siPB2-12 is SEQ ID No. 1 in the sequence listing, and the other strand sequence is SEQ ID No. 2 in the sequence listing; one strand sequence of siNP-4 is SEQ ID No. 5 in the sequence listing, and the other strand sequence is SEQ ID No. 6 in the sequence listing. The siPB2-12 and / or siNP-4 and their derivatives provided by this invention can effectively inhibit H1N1, H3N2, H5N6, H7N9, and H9N2.

Description

A broad-spectrum anti-influenza A virus siRNA molecule and its derivatives and their applications Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a broad-spectrum anti-influenza A virus siRNA molecule and its derivatives and their applications. Background Technology

[0002] Influenza virus (IV), belonging to the Orthomyxoviridae family, is an enveloped, single-stranded, negative-sense RNA virus. Influenza virus is the most common virus causing respiratory infections in humans, causing approximately 3 to 5 million illnesses and 290,000 to 650,000 deaths worldwide each year, demonstrating its severity.

[0003] Based on the antigenicity of their nucleocapside (NP) and matrix (M) proteins, influenza viruses can be classified into influenza A virus (IAV), influenza B virus (IBV), influenza C virus (ICV), and influenza D virus (IDV). Influenza A virus typically causes global pandemics; influenza B virus generally only causes localized outbreaks, but can also widely infect populations and cause severe cases; influenza C virus causes only mild upper respiratory tract infections and does not cause pandemics; currently, influenza D virus only infects cattle and pigs, not humans.

[0004] Antigenic mutations in influenza A viruses have given them the ability to evade host immune responses. The host's immune response to emerging variants may be weakened or even ineffective, leading to outbreaks of major and seasonal influenza. Due to mutations in influenza A viruses, antigenic drift and antigenic shifts occur in hemagglutinin (HA) and neuraminidase (NA), making novel influenza pandemics a constant possibility. Antiviral drugs are a common means of preventing influenza virus infection, but the number of existing antiviral drugs is limited. Furthermore, research data shows that the resistance rate of H1N1 subtype strains to oseltamivir is as high as 67%, and 193 out of 209 H3N2 subtype strains have developed resistance to amantadine. Moreover, highly pathogenic H5N1 avian influenza virus mutants resistant to oseltamivir have already emerged. In addition, the most effective method for treating and preventing influenza viruses is vaccination, but the development of vaccines to combat emerging novel influenza outbreaks is significantly delayed. Antigenic drift and antigenic shifts of the virus pose a constantly evolving challenge to available vaccines.

[0005] RNA interference (RNAi) is an important tool in gene function research and innovative drug development, primarily mediated by double-stranded small interfering RNAs (siRNAs) of the same length and reverse complementarity. siRNAs are 19–21 bp long, double-stranded complementary RNAs with a protruding base at the 3' end. They can be directly synthesized chemically or cleaved from dsRNA using the Dicer enzyme (a sequence-specific endonuclease). siRNA-mediated gene silencing disrupts the integrity of the target protein mRNA through complementary base pairing with the target RNA, effectively inhibiting the expression of disease-related genes and thus producing a therapeutic effect. siRNAs are characterized by high efficiency, strong gene targeting, short development cycles, abundant candidate targets, high therapeutic efficacy, low drug toxicity, and wide application areas, making them a valuable tool for in vivo and in vitro single-gene research and a novel approach for drug development. A paper titled "Asymmetric RNA duplexes mediate RNA interference in mammalian cells," published in *Nature Biotechnology* in December 2009, demonstrated that asymmetric siRNA (aiRNA) is a better option for gene silencing. The mechanism of action of aiRNA is the same as that of siRNA, but it typically contains a 15 bp (or less) sense strand, base overhangs at the 3' and 5' ends of the antisense strand, and an asymmetric structure where the sense strand is shorter than the antisense strand. Compared to siRNA, aiRNA has many significant advantages: ① RISC-dependent binding occurs between the antisense strand and the aiRNA, while the sense strand does not participate in the interference process, eliminating off-target effects. Furthermore, aiRNA's superior targeting specificity and high efficiency reduce the likelihood of toxic side effects due to off-target effects. ② aiRNA drugs exhibit high efficacy in silencing target genes, rapid onset of action, and long duration of action. ③ Experiments have demonstrated that aiRNA drugs can reduce or even eliminate the immunotoxicity of interferon responses. ④ The small molecular size and low non-specific interferon response of aiRNA simplify its delivery. ⑤ The short length of aiRNA eliminates the need for extensive chemical modifications, reducing drug development and synthesis costs. These advantages confirm that aiRNA is a better choice for gene silencing and represents a new hope for disease treatment.

[0006] In conclusion, given the limitations of current anti-influenza virus strategies, new strategies for the prevention and treatment of influenza viruses are urgently needed. Summary of the Invention

[0007] The technical problem to be solved by this invention is to provide siRNA capable of resisting multiple influenza subtypes. The technical problem to be solved is not limited to the described technical subject matter; other technical subject matter not mentioned herein will be clearly understood by those skilled in the art through the following description.

[0008] To solve the above-mentioned technical problems, the present invention provides the following technical solutions:

[0009] This invention provides a double-stranded RNA molecule, its modifications, or a pharmaceutically acceptable salt thereof, wherein the double-stranded RNA molecule is siPB2-12 and / or siNP-4, and / or a derivative thereof; one strand sequence of siPB2-12 is SEQ ID No. 1 in the sequence listing, and the other strand sequence is SEQ ID No. 2 in the sequence listing; one strand sequence of siNP-4 is SEQ ID No. 5 in the sequence listing, and the other strand sequence is SEQ ID No. 6 in the sequence listing.

[0010] The derivative of siPB2-12 is a double-linked RNA molecule named aiPB2-12, and the derivative of siNP-4 is a double-linked RNA molecule named aiNP-4. One strand of aiPB2-12 has the sequence of SEQ ID No. 3 in the sequence listing, and the other strand has the sequence of SEQ ID No. 4 in the sequence listing. One strand of aiNP-4 has the sequence of SEQ ID No. 7 in the sequence listing, and the other strand has the sequence of SEQ ID No. 8 in the sequence listing.

[0011] SEQ ID No. 1 and SEQ ID No. 2 both consist of 19 ribonucleotides. The ribonucleotides 1-19 of SEQ ID No. 1 are anticomplementary to those 1-19 of SEQ ID No. 2, and the resulting double-stranded RNA is named siPB2-12. SEQ ID No. 3 and SEQ ID No. 4 both consist of 15 ribonucleotides. The ribonucleotides 1-15 of SEQ ID No. 3 are anticomplementary to those 2-16 of SEQ ID No. 10, and the resulting double-stranded RNA is named siPB2-12. Nucleotides 4-18 of SEQ ID No. 3 are identical to those of SEQ ID No. 1, and SEQ ID No. 4 and SEQ ID No. 2 share the same target site, the difference being that the TT modification in SEQ ID No. 2 is at the 3' end, while in SEQ ID No. 4 it is at the 5' end. SEQ ID No. 5 and SEQ ID No. 6 are similar to SEQ ID No. 1 and SEQ ID No. 2. The same applies to SEQ ID No. 7 and SEQ ID No. 8 as to SEQ ID No. 3 and SEQ ID No. 4.

[0012] The term "modifier" refers to a product obtained by modifying the double-stranded RNA molecule. Various modification methods can be used, including one or more combinations selected from ribose modification, base modification, and phosphate backbone modification. The term "pharmaceutically acceptable salt" refers to a salt suitable for contact with human and lower animal tissues without excessive toxicity, irritation, or allergic reactions, within the bounds of reliable medical judgment, and with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, SMBerge, et al., J. Pharmaceutical Sciences, 1977, 66:1 provides a detailed description of pharmaceutically acceptable salts.

[0013] The present invention also provides a biological material containing the aforementioned double-stranded RNA molecule, wherein the biological material is a DNA molecule capable of producing the double-stranded RNA molecule, a recombinant vector containing the DNA molecule, or a recombinant microorganism containing the DNA molecule.

[0014] The present invention also provides an influenza virus inhibitor, wherein the influenza virus inhibitor contains the aforementioned double-stranded RNA molecule, its modifications, or a pharmaceutically acceptable salt thereof.

[0015] The present invention also provides a medicament for the prevention and / or treatment of influenza, said medicament containing the aforementioned double-stranded RNA molecule, its modifications, or a pharmaceutically acceptable salt thereof.

[0016] The influenza virus inhibitors or drugs described herein are available in dosage forms such as aerosols, tablets, capsules, drops, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, lozenges, suppositories, or lyophilized powder injections.

[0017] In the above text, the active ingredients of the influenza virus inhibitor and the medicament for preventing and / or treating influenza may be the double-stranded RNA molecule, its modifications, or a pharmaceutically acceptable salt thereof. The active ingredients of the influenza virus inhibitor and the medicament for preventing and / or treating influenza may also contain other substances, which can be determined by those skilled in the art based on the efficacy of the influenza virus inhibitor and the medicament for preventing and / or treating influenza.

[0018] In the above description, the influenza virus inhibitor and the drug for preventing and / or treating influenza, in addition to containing the double-stranded RNA molecule, its modifications, or a pharmaceutically acceptable salt thereof, may also contain a suitable carrier or excipient. The carrier materials here include, but are not limited to, water-soluble carrier materials (such as polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), and enteric-coated carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.). Water-soluble carrier materials are preferred. Using these materials, various dosage forms can be formulated, including but not limited to tablets, capsules, pellets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, lozenges, suppositories, lyophilized powder injections, etc. These can be conventional formulations, sustained-release formulations, controlled-release formulations, and various microparticle delivery systems. Various carriers known in the art can be widely used to formulate unit-dose dosage forms into tablets. Examples of carriers include diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, and aluminum silicate; humectants and binders such as water, glycerin, polyethylene glycol, ethanol, propanol, starch paste, dextrin, syrup, honey, glucose solution, gum arabic paste, gelatin paste, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, and polyvinylpyrrolidone; and disintegrants. Examples of carriers include dried starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitol fatty acid esters, sodium dodecyl sulfate, methylcellulose, and ethylcellulose; disintegration inhibitors include sucrose, tristearate, cocoa butter, and hydrogenated oil; absorption enhancers include quaternary ammonium salts and sodium dodecyl sulfate; and lubricants include talc, silica, corn starch, stearates, boric acid, liquid paraffin, and polyethylene glycol. Tablets can also be further formulated into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer and multilayer tablets. Various carriers known in the art can be widely used to formulate unit-dose dosage forms into pills. Examples of carriers include diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, gelucire, kaolin, and talc; binders such as gum arabic, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste, or flour paste; and disintegrants such as agar powder, dried starch, alginate, sodium dodecyl sulfate, methylcellulose, and ethylcellulose. Various carriers known in the art can be widely used to formulate unit dosage forms into suppositories. Examples of carriers include polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, and semi-synthetic glycerides.To formulate unit-dose dosage forms for injection, such as solutions, emulsions, lyophilized powders for injection, and suspensions, all diluents commonly used in the art can be used, such as water, ethanol, polyethylene glycol, 1,3-propanediol, ethoxylated isostearyl alcohol, polyoxyethylene isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters, etc. Additionally, to prepare isotonic injections, appropriate amounts of sodium chloride, glucose, or glycerol can be added to the injection formulation. Furthermore, conventional solubilizers, buffers, pH adjusters, etc., can also be added. Furthermore, if necessary, colorants, preservatives, flavorings, tasters, sweeteners, or other materials can be added to the pharmaceutical formulation. The above dosage forms can be administered via injection, including subcutaneous, intravenous, intramuscular, and intracavitary injections; via cavities, such as rectal and vaginal; via the respiratory tract, such as nasal; and via mucosal administration. Injection is the preferred route of administration.

[0019] The present invention also provides the use of the aforementioned double-stranded RNA molecule, its modifications, or pharmaceutically acceptable salts thereof, or the aforementioned biological materials in the preparation of influenza virus inhibitors or in the inhibition of influenza viruses.

[0020] Specifically, the influenza virus is H1N1, H3N2, H5N6, and / or H9N2 virus, and the influenza is caused by H1N1, H3N2, H5N6, H7N9, and / or H9N2 virus. The H1N1, H3N2, H5N6, H7N9, and / or H9N2 viruses specifically originate from A / Guangdong-Maonan / SWL1536 / 2019 (H1N1), A / Hong Kong / 2671 / 2019 (H3N2), A / Guangzhou / 39715 / 2014 (H5N6), A / Anhui / 1 / 2013 (H7N9), and A / Guangxi / NN10.19T-NGS / 2018 (H9N2).

[0021] This invention demonstrates through specific experiments that siPB2-12, designed based on the influenza A gene, and aiPB2-12, designed based on siPB2-12, can effectively inhibit H1N1, H3N2, H5N6, H7N9, and H9N2, providing a new strategy for the prevention and treatment of influenza viruses. Attached Figure Description

[0022] Figure 1 shows the hemagglutination titer (HA) in the supernatant of MDCK cells infected with A / Guangdong-Maonan / SWL1536 / 2019(H1N1). MDCK cells were transfected with siRNAs targeting the (a)PB2, (b)PB1, (c)PA, and (d)NP genes, respectively. The hemagglutination titer in the cell supernatant was measured 48 h post-infection. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

[0023] Figure 2 shows the Western blot analysis results of NP protein in cell supernatant 48 h after infection with A / Guangdong-Maonan / SWL1536 / 2019(H1N1).

[0024] Figure 3 shows the Western blot analysis results of intracellular HA and NP proteins 48 h after infection with A / Guangdong-Maonan / SWL1536 / 2019(H1N1).

[0025] Figure 4 shows the determination of HA titer in the cell supernatant of MDCK cells infected with different subtypes of influenza virus. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

[0026] Figure 5 shows the results of Western blot analysis of NP protein in cell supernatant 48 h after infection with different subtypes of influenza virus.

[0027] Figure 6 shows the Western blot analysis results of intracellular NP proteins 48 hours after infection with different subtypes of influenza virus.

[0028] Figure 7 shows the determination of influenza virus HA titer in cell supernatant by aiRNA interference. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

[0029] Figure 8 shows the results of Western blot analysis of influenza virus NP protein in cell supernatant by aiRNA interference.

[0030] Figure 9 shows the results of Western blot analysis of aiRNA interfering with the intracellular influenza virus NP protein. Detailed Implementation

[0031] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0032] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0033] Anti-NP antibody: The rabbit polyclonal antibody prepared by immunizing rabbits with the NP protein of A / WSN / 1993(H1N1) (amino acid sequence number QDQ43414.1 on the NCBI website) is described in the literature "Neddylation of M1 negatively regulates the replication of influenza A virus".

[0034] Anti-HA antibody: The HA primary antibody is a rabbit-derived polyclonal antibody immunized with baculovirus-expressed H1 protein (amino acid sequence number ACQ76318.1 on the NCBI website). It was prepared and preserved by the inventor's laboratory and is described in the literature "Avian-to-HumanReceptor-Binding Adaptation of Avian H7N9 Influenza Virus Hemagglutinin".

[0035] The following examples used GraphPad Prism 8 statistical software to process the data and employed a two-way ANOVA test. P < 0.05 indicates a significant difference.

[0036] Example 1: Initial screening of candidate siRNA molecules

[0037] siRNA sequences were designed targeting the PB2, PB1, PA, and NP genes of influenza A virus. The siRNA molecules used in this embodiment are shown in Table 1, and the aiRNA molecules are also shown in Table 1. The siRNA sequences were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0038] Table 1 Candidate siRNA molecules

[0039]

[0040] The first 19th bits of the Sense strand of siPB2-12 correspond to SEQ ID No. 1, and the first 19th bits of the Sense strand of siPB2-12 correspond to SEQ ID No. 2.

[0041] The first 19th positions of the Sense strand of siNP-4 correspond to SEQ ID No. 5, and the first 19th positions of the Sense strand of siNP-4 correspond to SEQ ID No. 6.

[0042] 1. Experimental Methods

[0043] (1) Preparation of A / Guangdong-Maonan / SWL1536 / 2019(H1N1) virus

[0044] The experiment was conducted in the BSL-2 laboratory of the Institute of Microbiology, Chinese Academy of Sciences.

[0045] 100 μl of primary A / Guangdong-Maonan / SWL1536 / 2019(H1N1) virus was injected into the allantoic cavity of 10-day-old chicken embryos. After sealing with wax, the embryos were incubated at 37°C for 72 hours, and the growth status of the chicken embryos was observed every 12 hours. After 72 hours, the allantoic fluid was harvested and the hemagglutination titer was determined. The harvested allantoic fluid was the egg P1 generation A / Guangdong-Maonan / SWL 1536 / 2019(H1N1) virus.

[0046] (2) Cell culture and liposome transfection

[0047] MDCK cells were charged at 2.5 × 10⁻⁶ 6 Seed cells at a density of / wells into 6-well cell culture plates. When cells have adhered to the plates to 70-80% confluence, group them as follows:

[0048] siPB2-1 group: according to siRNA transfection reagent (purchased from: Invitrogen) TM (Product No.: 13778150) Instructions: Transfect siPB2-1 into MDCK cells using the siRNA transfection reagent Lipofectamine RNAiMAX. The final concentration of siRNA in each well is 50 nM. The experiment is repeated three times, with three replicate wells each time. After incubating the cell culture plate at 37°C and 5% CO2 for 24 h, aspirate the culture medium and wash three times with PBS. Infect cells with A / Guangdong-Maonan / SWL1536 / 2019 (H1N1) virus at an MOI of 0.1 and incubate at 37°C for 1 h. To promote viral infection, gently shake the cell culture plate every 15 min during incubation. After 1 h, aspirate the virus dilution and add 500 μl of virus maintenance medium (DMEM medium containing 2 μg / mL trypsin, 100 U / mL penicillin, and 0.1 mg / mL streptomycin) to each well. Continue incubation at 37°C and detect various indicators according to the following procedures:

[0049] ① Hemagglutination assay: Cell supernatant was harvested at 12h, 24h, 36h, and 48h post-infection. The hemagglutination titer of the influenza virus was determined by utilizing its ability to agglutinate red blood cells. The specific method for determining the hemagglutination titer of the virus is as follows: A 96-well V-type microplate was placed horizontally. Except for the first column, 25μl of PBS was added to each well. 50μl of the test virus solution (the aforementioned cell supernatant) was added to the first well of each column. Starting from the first well, the test virus solution was serially diluted up to the 12th well. 25μl of the last well was discarded. Then, 25μl of 1% chicken red blood cell suspension was added to each well. The plate was incubated at room temperature for 30min, and the presence of a hemagglutination reaction was observed. HA titer (log2) = log2N, where N is the dilution factor of the test virus solution.

[0050] ②Western-blot: 48 h after viral infection, the culture medium was aspirated to obtain the supernatant of cells transfected with siPB2-1 and infected with the H1N1 strain. The remaining adherent cells were washed once with PBS, and 100 μl of cell lysis buffer (prepared with DAPI lysis buffer and protease inhibitor PMSF at a ratio of 100:1) was added. The cells were incubated at 4°C for 10 min. Cells were then pipetted to transfer as much of the resulting cell lysis buffer as possible into a new 1.5 ml EP tube. The tubes were centrifuged at 4°C and 12000 rpm for 5 min. The supernatant was mixed with SDS-PAGE Loading Buffer (5×) at a ratio of 4:1. After a brief centrifugation, the diluted cell sample was boiled at 100°C for 5–10 min and stored at -80°C for later use. The expression of viral NP protein, HA protein, and experimental internal control β-actin protein was detected by Western blot. The antibodies used for detection included anti-NP antibody, anti-HA antibody, and anti-β-actin antibody (purchased from Proteintech, catalog number: 20536-1-AP).

[0051] The following groups are listed: siPB2-2, siPB2-11, siPB2-12, siPB1-1, siPB1-2, siPB1-3, siPB1-4, siPB1-5, siPB1-6, siPA-1, siPA-2, siPA-3, siPA-4, siNP-1, siNP-2, siNP-3, siNP-4, siNP-5, siNP-6, siNP-7, and siNP-8: (The last part, "siPB2," appears to be a typo and can be left as is.) -2, siPB2-11, siPB2-12, siPB1-1, siPB1-2, siPB1-3, siPB1-4, siPB1-5, siPB1-6, siPA-1, siPA-2, siPA-3, siPA-4, siNP-1, siNP-2, siNP-3, siNP-4, siNP-5, siNP-6, siNP-7 or siNP-8 replace siPB2-1 in group siPB2-1, and the remaining operations are the same as group siPB2-1.

[0052] siNC: Replace siPB2-1 in group siPB2-1 with siNC, and perform the other operations as in group siPB2-1.

[0053] siGFP: Replace siPB2-1 in group siPB2-1 with siGFP, and perform the other operations as in group siPB2-1.

[0054] MOCK: Replace the siRNA and transfection reagent complex in the siPB2-1 group with Lipofectamine RNAiMAX, and perform the other operations as in the siPB2-1 group.

[0055] Control (virus): Replace the siRNA transfection reagents Lipofectamine RNAiMAX and siPB2-1 in the siPB2-1 group with DMEM medium, and perform the other operations as in the siPB2-1 group.

[0056] 2. Results

[0057] (1) Determination of blood coagulation titer

[0058] Compared with the control group, (a) the HA titer in the cell supernatant of the siPB2-1 and siPB2-11 transfected groups showed a decreasing trend, but the difference was not statistically significant; the HA titer in the cell supernatant of the siPB2-2 and siPB2-12 transfected groups was significantly decreased (P < 0.001 and P < 0.01, respectively), and the titer of the three replicates in the siPB2-2 group was 2. 1 2 1 2 2siPB 2-12 groups have three replicates with 2 2 2 1 2 3 (b) The HA titer in the supernatant of cells transfected with siPB1-4 and siPB1-6 showed a decreasing trend, but the difference was not statistically significant. (c) The HA titer in the supernatant of cells transfected with siPA-1, siPA-3, and siPA-4 showed a decreasing trend, but the difference was not statistically significant; however, the HA titer in the supernatant of cells transfected with siPA-2 was significantly reduced (P < 0.01). (d) The HA titer in the supernatant of cells transfected with siNP-2, siNP-3, siNP-4, siNP-5, siNP-6, siNP-7, and siNP-8 showed a decreasing trend, but the difference was not statistically significant.

[0059] Therefore, compared with the control group, transfection with siPB2-2, siPB2-12, and siPA-2 can significantly inhibit the replication of A / Guangdong-Maonan / SWL1536 / 2019(H1N1) virus in MDCK cells (Figure 1).

[0060] (2) Detection of NP protein in cell supernatant

[0061] Inhibition effect = (Control histone band gray value - Transfected histone band gray value) / Control histone band gray value × 100%. The gray value statistical rules are as follows: the gray values ​​are obtained by analyzing the protein bands in the Western blot results using Image software.

[0062] At 48 h after viral infection, the expression of influenza virus NP protein in the cell supernatant was analyzed by Western blot (see Figure 2). siRNA showed that it had a certain inhibitory effect on the synthesis of influenza virus NP protein in the cell supernatant. Compared with the Control group, in the siRNA-PB2 transfection group, siPB2-1, siPB2-2, and siPB2-11 showed inhibitory effects of 59%, 45%, and 49% on NP protein, respectively, with siPB2-12 showing the best inhibitory effect at 78%. In the siRNA-PB1 transfection group, siPB1-3 and siPB1-4 showed inhibitory effects of 14% and 20% on NP protein, respectively, while the remaining siRNA-PB1 showed no inhibitory effect or only a slight inhibitory effect. In the siRNA-PA transfection group, siPA-2 and siPA-4 showed inhibitory effects of 23% and 12% on NP protein, respectively, while the remaining siRNA-PA showed no inhibitory effect or only a slight inhibitory effect. In the siRNA-NP transfection group, siNP-4 showed an inhibitory effect of 25% on NP protein, while the remaining siRNA-NP showed no inhibitory effect or only a slight inhibitory effect.

[0063] (3) Detection of intracellular NP and HA proteins

[0064] The method for calculating the inhibition effect is referenced in (2).

[0065] At 48 h after viral infection, the expression of influenza virus NP and HA proteins in cells was analyzed by Western blot (see Figure 3). siRNA showed a certain inhibitory effect on the synthesis of influenza virus NP and HA proteins in cells. Compared with the control group, in the siRNA-PB2 transfection group, siPB2-1, siPB2-2, and siPB2-12 showed inhibitory effects of 27%, 21%, and 38% on NP protein, and 62%, 54%, and 80% on HA protein, respectively. In the siRNA-PB1 transfection group, siPB1-1, siPB1-2, siPB1-3, siPB1-4, siPB1-5, and siPB1-6 showed inhibitory effects of 49%, 32%, 24%, 36%, 17%, and 10% on HA protein, respectively, but no or minimal inhibitory effect on NA protein. In the siRNA-PA transfection group, siPA-1, siPA-2, siPA-3, and siPA- The inhibitory effects of siNP-2, siNP-3, siNP-4, siNP-5, siNP-6, siNP-7, and siNP-8 on NP protein were 28%, 61%, 48%, and 37%, respectively, and on HA protein, respectively. In the siRNA-NP transfection group, the inhibitory effects of siNP-2, siNP-3, siNP-4, siNP-5, siNP-6, siNP-7, and siNP-8 on NP protein were 46%, 56%, 73%, 60%, 55%, 49%, and 47%, respectively, while the inhibitory effects of siNP-1, siNP-2, siNP-3, siNP-4, siNP-5, siNP-6, siNP-7, and siNP-8 on NP protein were 46%, 57%, 73%, 74%, 46%, 48%, 49%, and 35%, respectively.

[0066] Example 2: Verification of siRNA interference function against different subtypes of influenza virus

[0067] A / Guangdong-Maonan / SWL1536 / 2019(H1N1): Described in the literature "The determination of haemagglutinin influenza antibodies in the Polish population in the epidemic season 2020 / 2021 during the SARS-CoV-2 pandemic", it is available to the public from the Institute of Microbiology, Chinese Academy of Sciences. This biological material is only for repeating the relevant experiments of this invention and should not be used for other purposes.

[0068] A / Hong Kong / 2671 / 2019(H3N2): Described in the literature “The determination of haemagglutinin influenza antibodies in the Polish population in the epidemic season 2020 / 2021 during the SARS-CoV-2 pandemic”, this biological material is available to the public from the Institute of Microbiology, Chinese Academy of Sciences. This biological material is only for repeating the relevant experiments of this invention and should not be used for other purposes.

[0069] A / Guangzhou / 39715 / 2014(H5N6): Described in the document "The complexity of human-infected AIV H5N6 isolated from China", it is available to the public from the Institute of Microbiology, Chinese Academy of Sciences. This biological material is only for repeating the relevant experiments of this invention and should not be used for other purposes.

[0070] A / Anhui / 2013(H7N9): Described in the literature “Differences in the pathogenicity and inflammation response induced by avian influenza A H7N9 virus infection in BALBc and C57BL6 mouse models”, this biological material is available to the public from the Institute of Microbiology, Chinese Academy of Sciences. This biological material is only for repeating the relevant experiments of this invention and should not be used for other purposes.

[0071] A / Guangxi / NN10.19T-NGS / 2018(H9N2): Described in the literature “Genetic, biological and epidemiological study on a cluster of H9N2 avian influenza virus infections among chick ens, a pet cat, and humans at a backyard farm in Guangxi, China”, this biological material is available to the public from the Institute of Microbiology, Chinese Academy of Sciences. This biological material is only for repeating the relevant experiments of this invention and should not be used for other purposes.

[0072] Based on the results of Example 1, siPB2-12, siPB1-4, siPA-2, and siNP-4 from the candidate siRNA molecules in Example 1 were selected for broad-spectrum anti-influenza virus screening on strains A / Guangdong-Maonan / SWL1536 / 2019(H1N1), A / Hong Kong / 2671 / 2019(H3N2), A / Guangzhou / 39715 / 2014(H5N6), A / Anhui / 2013(H7N9), and A / Guangxi / NN 10.19T-NGS / 2018(H9N2). The preparation methods for Kong / 2671 / 2019(H3N2), A / Guangzhou / 39715 / 2014(H5N6), A / Anhui / 2013(H7N9), and A / Guangxi / NN10.19T-NGS / 2018(H9N2) were all as described in Example 1. siRNA transfection and virus inoculation were performed according to the experimental methods shown in Example 1.

[0073] (1) Determination of blood coagulation titer

[0074] MDCK cells were transfected with siPB2-12, siPB1-4, siPA-2, and siNP-4, respectively, with siRNA-NC, siRNA-GFP, and Control (virus infection only) serving as negative controls. Twenty-four hours after transfection, MDCK cells were infected with influenza virus strains A / Guangdong-Maonan / SWL1536 / 2019 (H1N1), A / Hong Kong / 2671 / 2019 (H3N2), A / Guangzhou / 39715 / 2014 (H5N6), A / Anhui / 1 / 2013 (H7N9), and A / Guangxi / NN10.19T-NGS / 2018 (H9N2), respectively. Cell supernatants were harvested 48 hours after virus infection, and the hemagglutination titer of the virus in the supernatant was measured (see Figure 4).

[0075] The results showed that, compared with the control group, HA titer in the supernatant of siPB2-12-targeted A / Guangdong-Maonan / SWL1536 / 2019(H1N1), A / Hong Kong / 2671 / 2019(H3N2), A / Guangzhou / 39715 / 2014(H5N6), and A / Guangxi / NN10.19T-NGS / 2018(H9N2) cells was significantly reduced (P<0.0001, P<0.001, P<0.001, P<0.05, respectively); siPB1-4-targeted A / Guangdong-Maonan / SWL1536 / 2019(H1N1), A / Hong Kong / 2671 / 2019(H3N2), A / Guangzhou / 39715 / 2014(H5N6), and A / Guangxi / NN10.19T-NGS / 2018(H9N2) cells was significantly reduced (P<0.0001, P<0.001, P<0.001, P<0.05, respectively). The HA titer in the supernatant of Kong / 2671 / 2019(H3N2), A / Guangzhou / 39715 / 2014(H5N6), A / Anhui / 1 / 2013(H7N9), and A / Guangxi / NN10.19T-NGS / 2018(H9N2) cells was significantly decreased (P<0.01, P<0.0001, P<0.0001, P<0.01, and P<0.05, respectively); siPA-2 targeting A / Anhui / 1 / 2013(H7N9) cells significantly decreased HA titer (P<0.01), targeting A / Hong A decrease in HAtiter in the supernatant of cells from Kong / 2671 / 2019(H3N2), A / Guangzhou / 39715 / 2014(H5N6), and A / Guangxi / NN10.19T-NGS / 2018(H9N2) was observed, but this was not statistically significant. siNP-4 targeting HAtiter in the supernatant of cells from A / Guangdong-Maonan / SWL1536 / 2019(H1N1), A / Hong Kong / 2671 / 2019(H3N2), A / Guangzhou / 39715 / 2014(H5N6), A / Anhui / 1 / 2013(H7N9), and A / Guangxi / NN10.19T-NGS / 2018(H9N2) was also observed. titer decreased significantly (P < 0.01, P < 0.001, P < 0.001, P < 0.01 and P < 0.05, respectively).

[0076] When siPB2-12 targeted A / Guangdong-Maonan / SWL1536 / 2019(H1N1), the titer in the three replicates in this group was 2. 1 2 1 2 2When siPB2-12 targets A / Hong Kong / 2671 / 2019(H3N2), the titer of the three replicates in this group is 2. 1 2 0 2 0 When siPB2-12 was targeted at A / Guangzhou / 39715 / 2014(H5N6), the titer in the three replicates in this group was 2. 6 2 6 2 6 When siPB2-12 targets A / Guangxi / NN10.19T-NGS / 2018(H9N2), the valence of the three replicates in this group is 2. 0 2 0 2 0 .

[0077] When siPB1-4 targets A / Guangdong-Maonan / SWL1536 / 2019(H1N1), the titer in the three replicates in this group is 2. 2 2 2 2 2 When siPB1-4 targets A / Hong Kong / 2671 / 2019(H3N2), the titer of the three replicates in this group is 2. 0 2 0 2 0 When siPB1-4 targets A / Guangzhou / 39715 / 2014(H5N6), the titer in the three replicates in this group is 2. 6 2 6 2 6 When siPB1-4 targets A / Guangxi / NN10.19T-NGS / 2018(H9N2), the titer of the three replicates in this group is 2. 0 2 0 2 0 .

[0078] When siNP-4 targets A / Guangdong-Maonan / SWL1536 / 2019(H1N1), the titer in the three replicates in this group is 2. 2 2 2 2 2 When siNP-4 targets A / Hong Kong / 2671 / 2019(H3N2), the titer of the three replicates in this group is 2. 1 2 0 20 When siNP-4 was targeted at A / Guangzhou / 39715 / 2014(H5N6), the titer in the three replicates in this group was 2. 6 2 6 2 6 When siNP-4 targets A / Guangxi / NN10.19T-NGS / 2018(H9N2), the titer of the three replicates in this group is 2. 0 2 0 2 1 .

[0079] (2) Detection of NP protein in cell supernatant

[0080] At 48 h after viral infection, the expression of influenza virus NP protein in cell supernatant was analyzed by Western blot (see Figure 5). siRNA showed a certain inhibitory effect on the synthesis of influenza virus NP protein in cell supernatant. Compared with the control group, siPB2-12 targeting NP protein in the supernatant of A / Guangdong-Maonan / SWL1536 / 2019(H1N1), A / Hong Kong / 2671 / 2019(H3N2), A / Guangzhou / 39715 / 2014(H5N6), A / Anhui / 1 / 2013(H7N9), and A / Guangxi / NN10.19T-NGS / 2018(H9N2) cells showed inhibitory effects of 61%, 48%, 46%, 31%, and 43%, respectively. siPB1-4 targeting A / Hong The inhibitory effects of siPA-2 on NP protein in the supernatant of Kong / 2671 / 2019(H3N2), A / Guangzhou / 39715 / 2014(H5N6), and A / Guangxi / NN10.19T-NGS / 2018(H9N2) cells were 44%, 53%, and 59%, respectively; siPA-2 targeting A / Guangdong-Maonan / SWL1536 / 2019(H1N1), A / Guangzhou / 39715 / 2014(H5N6), A / Anhui / 1 / 2013(H7N9), and A / Guangxi / The inhibitory effects of siNP-4 on NP protein in the supernatant of NN10.19T-NGS / 2018(H9N2) cells were 62%, 40%, 36%, and 20%, respectively; the inhibitory effects of siNP-4 on NP protein in the supernatant of A / Guangdong-Maonan / SWL1536 / 2019(H1N1), A / Guangzhou / 39715 / 2014(H5N6), A / Anhui / 1 / 2013(H7N9), and A / Guangxi / NN10.19T-NGS / 2018(H9N2) cells were 84%, 41%, 40%, and 21%, respectively.

[0081] (3) Detection of intracellular NP proteins

[0082] At 48 h after viral infection, the expression of influenza virus NP protein in cells was analyzed by Western blot (see Figure 6). siRNA showed a certain inhibitory effect on the synthesis of influenza virus NP protein in cells. Compared with the control group, siPB2-12 targeting intracellular NP protein of A / Guangdong-Maonan / SWL1536 / 2019(H1N1), A / HongKong / 2671 / 2019(H3N2), A / Guangzhou / 39715 / 2014(H5N6), and A / Guangxi / NN10.19T-NGS / 2018(H9N2) cells showed inhibitory effects of 60%, 68%, 39%, and 65%, respectively; siPB1-4 targeting A / Guangdong-Maonan / SWL1536 / 2019(H1N1), A / HongKong / 2671 / 2019(H3N2), and A / HongKong / 2671 / 2019(H3N2) cells showed inhibitory effects of 65%, 68%, 39%, and 65%, respectively. The inhibitory effects of siPA-2 on intracellular NP proteins in cells of A / Guangdong-Maonan / SWL1536 / 2019(H1N1), A / Hong Kong / 2671 / 2019(H3N2), and A / Guangzhou / 39715 / 2014(H5N6) were 12%, 44%, 11%, and 48%, respectively; the inhibitory effects of siPA-2 on intracellular NP proteins in cells of A / Guangdong-Maonan / SWL1536 / 2019(H1N1), A / Hong Kong / 2671 / 2019(H3N2), and A / Guangzhou / 39715 / 2014(H5N6) were 46%, 42%, and 24%, respectively; the inhibitory effects of siNP-4 on intracellular NP proteins in cells of A / Guangdong-Maonan / SWL1536 / 2019(H1N1), A / Hong Kong / 2671 / 2019(H3N2), and A / Guangzhou / 39715 / 2018(H9N2) were 46%, 42%, and 24%, respectively; The inhibitory effects on intracellular NP protein in Kong / 2671 / 2019(H3N2) cells were 51% and 36%, respectively.

[0083] In summary, siPB2-12 and siNP-4 can broadly inhibit H1, H3, H5, H7 and H9 subtype influenza viruses, demonstrating broad-spectrum activity.

[0084] Example 3: Verification of the interference function of aiRNA against different subtypes of influenza virus

[0085] After initial screening of candidate siRNA molecules, four siRNAs—siPB2-12, siPB1-4, siPA-2, and siNP-4—were selected, and their positive strands were shortened to 15 bp, as detailed below:

[0086] Table 2 aiPB2 molecules

[0087] siRNASensestrand(5'-3')Antisensestrand(5'-3')aiPB2-12CUCUAGCAUACUUACTTAGUAAGUAUGCUAGAGUCCaiPB1-4UGAUGGGCAUGUUCATTUUGAACAUGCCCAUCAUCAaiPA-2UGGUUCAACUCCUUCTTGGAAGGAGUUGAACCAAGAaiNP-4UUUCUUCGGAGACAATTAUUGUCUCCGAAGAAAUAA surface

[0088] The first 15 bits of the Sense strand of aiPB2-12 correspond to SEQ ID No. 3, and the second 21 bits of the Sense strand of aiPB2-12 correspond to SEQ ID No. 4.

[0089] The first 15 positions of the sense strand of aiNP-4 correspond to SEQ ID No. 7, and the second 21 positions of the sense strand of aiNP-4 correspond to SEQ ID No. 8.

[0090] The specific screening method is as follows:

[0091] 1. Preparation of A / Guangdong-Maonan / SWL1536 / 2019(H1N1) virus

[0092] After MDCK cells were cultured in culture flasks until a monolayer was formed, primary A / Guangdong-Maonan / SWL1536 / 2019(H1N1) virus (MOI = 0.1) was inoculated onto the MDCK cells and incubated at 35°C for 72 h. The virus supernatant and cell debris were collected by shaking the cell culture flasks. After centrifugation, the supernatant was collected, aliquoted, and stored at -80°C for three freeze-thaw cycles to obtain A / Guangdong-Maonan / SWL1536 / 2019(H1N1) virus.

[0093] 2. Cell culture and liposome transfection

[0094] MDCK cells were charged at 2.5 × 10⁻⁶ 6 Seed cells at a density of / wells into 6-well cell culture plates. When cells have adhered to the plates to 70-80% confluence, group them as follows:

[0095] Group aiPB2-12: Transfected with siRNA using Invitrogen reagent (purchased from Invitrogen). TM(Product No.: 13778150) Instructions: Transfect aiPB2-12 cells into MDCK cells using the siRNA transfection reagent Lipofectamine RNAiMAX. The final concentration of siRNA in each well is 50 nM. The experiment is repeated three times, with three replicate wells each time. After incubating the cell culture plate at 37°C and 5% CO2 for 24 h, the culture medium is aspirated and the cells are washed three times with PBS. Cells are infected with A / Guangdong-Maonan / SWL1536 / 2019 (H1N1) virus at an MOI of 0.1 and incubated at 37°C for 1 h. To promote viral infection, the cell culture plate is gently shaken every 15 min during incubation. After 1 h, the virus dilution is aspirated, and 500 μl of virus maintenance medium (DMEM medium containing 2 μg / mL trypsin, 100 U / mL penicillin, and 0.1 mg / mL streptomycin) is added to each well. The cells are then incubated at 37°C, and the following parameters are measured:

[0096] ① Hemagglutination assay: Cell supernatant was harvested at 12h, 24h, 36h, and 48h post-infection. The hemagglutination titer of the influenza virus was determined by utilizing its ability to agglutinate red blood cells. The specific method for determining the hemagglutination titer of the virus is as follows: A 96-well V-type microplate was placed horizontally. Except for the first column, 25μl of physiological saline was added to each well. 50μl of the test virus solution (the aforementioned cell supernatant) was added to the first well of each column. Starting from the first well, the test virus solution was serially diluted up to the 12th well. 25μl of the last well was discarded. Then, 25μl of 1% chicken red blood cell suspension was added to each well. The plate was incubated at room temperature for 30min, and the presence of a hemagglutination reaction was observed. HA titer (log2) = log2N, where N is the dilution factor of the test virus solution.

[0097] ②Western-blot: 48 h after viral infection, the culture medium was aspirated to obtain the supernatant of cells transfected with siPB2-1 and infected with the H1N1 strain. The remaining adherent cells were washed once with PBS, and 100 μl of cell lysis buffer (prepared with DAPI lysis buffer and protease inhibitor PMSF at a ratio of 100:1) was added. The cells were incubated at 4°C for 10 min, and the cells were pipetted to transfer as much of the resulting cell lysis buffer as possible into a new 1.5 ml EP tube. The cells were centrifuged at 4°C and 12000 rpm for 5 min. The supernatant was mixed with SDS-PAGE Loading Buffer (5×) at a ratio of 4:1. After a brief centrifugation, the diluted cell sample was boiled at 100°C for 5–10 min and stored at -80°C for later use. Western blot was used to detect the expression of viral NP protein, HA protein, and the experimental internal control β-actin protein. The antibodies used for detection included anti-NP antibody, anti-HA antibody, and anti-β-actin antibody.

[0098] For the siPB2-12 group, aiPB1-4 group, siPB1-4 group, aiPA-2 group, siPA-2 group, aiNP-4 group, or siNP-4 group: replace aiPB2-12 in the aiPB2-12 group with the siPB2-12, aiPB1-4, siPB1-4, aiPA-2, siPA-2 group, aiNP-4, or siNP-4 siRNA (or aiRNA), and perform the other operations as in the aiPB2-12 group.

[0099] Control (virus) group: replace the siRNA transfection reagents Lipofectamine RNAiMAX and aiPB2-12 in the siPB2-1 group with pure DMEM medium, and the rest of the operation is the same as the aiPB2-12 group.

[0100] The experimental results are as follows:

[0101] (1) Determination of blood coagulation titer

[0102] MDCK cells were transfected with siPB2-12 / aiPB2-12, siPB1-4 / aiPB1-4, siPA-2 / aiPA-2, and siNP-4 / aiNP-4, respectively, with siRNA-GFP and Control (virus infection only) serving as negative controls. Twenty-four hours after transfection, MDCK cells were infected with influenza virus strains A / Guangdong-Maonan / SWL1536 / 2019 (H1N1), A / Hong Kong / 2671 / 2019 (H3N2), A / Guangzhou / 39715 / 2014 (H5N6), A / Anhui / 1 / 2013 (H7N9), and A / Guangxi / NN10.19T-NGS / 2018 (H9N2), respectively. Cell supernatants were harvested 48 hours after virus infection, and the hemagglutination titer of the virus in the supernatant was detected (see Figure 7).The results showed that compared with the control group, aiPB2-12 significantly reduced HA titer in the supernatant of cells targeting A / Guangdong-Maonan / SWL1536 / 2019(H1N1), A / Hong Kong / 2671 / 2019(H3N2), A / Guangzhou / 39715 / 2014(H5N6), and A / Anhui / 1 / 2013(H7N9) (P < 0.0001, P < 0.0001, P < 0.0001, respectively). AiPB1-4 targeting A / Hong Kong / 2671 / 2019(H3N2) cells showed a decreasing trend in HA titer, but this was not statistically significant. HA titer was significantly decreased in the supernatant of cells from Kong / 2671 / 2019(H3N2), A / Guangzhou / 39715 / 2014(H5N6), and A / Anhui / 1 / 2013(H7N9) (P<0.001, P<0.0001, P<0.0001, respectively). A decreasing trend in HA titer was observed in the supernatant of cells targeting A / Guangxi / NN10.19T-NGS / 2018(H9N2), but this was not statistically significant. aiPA-2 targeting HA titer in the supernatant of cells from A / Hong Kong / 2671 / 2019(H3N2), A / Guangzhou / 39715 / 2014(H5N6), and A / Anhui / 1 / 2013(H7N9) also showed a decrease. HA titer was significantly reduced (P < 0.001, P < 0.0001, P < 0.0001, respectively). A decreasing trend was observed in the supernatant of cells targeting A / Guangdong-Maonan / SWL1536 / 2019(H1N1) and A / Guangxi / NN10.19T-NGS / 2018(H9N2), but this was not statistically significant. aiNP-4 targeting HA titer in the supernatant of cells targeting A / Guangdong-Maonan / SWL1536 / 2019(H1N1), A / Hong Kong / 2671 / 2019(H3N2), A / Guangzhou / 39715 / 2014(H5N6), A / Anhui / 1 / 2013(H7N9), and A / Guangxi / NN10.19T-NGS / 2018(H9N2) also showed a decrease. titer decreased significantly (P < 0.0001, P < 0.01, P < 0.0001, P < 0.0001, P < 0.01, respectively).

[0103] When aiPB2-12 targeted A / Guangdong-Maonan / SWL1536 / 2019(H1N1), the titer in the three replicates in this group was 2.0 2 0 2 0 When aiPB2-12 targeted A / Hong Kong / 2671 / 2019(H3N2), the titer in the three replicates in this group was 2. 4 2 4 2 4 When aiPB2-12 targeted A / Guangzhou / 39715 / 2014(H5N6), the titer in the three replicates in this group was 2. 4 2 4 2 4 When aiPB2-12 targeted A / Anhui / 1 / 2013(H7N9), the titer in the three replicates in this group was 2. 5 2 5 2 5 When aiPB2-12 targets A / Guangxi / NN10.19T-NGS / 2018(H9N2), the valence of the three replicates in this group is 2. 1 2 1 2 2 .

[0104] When aiNP-4 targets A / Guangdong-Maonan / SWL1536 / 2019(H1N1), the titer in the three replicates in this group is 2. 0 2 0 2 0 When aiNP-4 targeted A / Hong Kong / 2671 / 2019(H3N2), the titer in the three replicates in this group was 2. 4 2 4 2 4 When aiNP-4 was targeted at A / Guangzhou / 39715 / 2014(H5N6), the titer in the three replicates in this group was 2. 4 2 4 2 4 When aiNP-4 was targeted at A / Anhui / 1 / 2013(H7N9), the titer in the three replicates in this group was 2. 5 2 5 2 5 When aiNP-4 targets A / Guangxi / NN10.19T-NGS / 2018(H9N2), the valence of the three replicates in this group is 2. 1 2 1 2 0 .

[0105] Therefore, compared with the control group, transfection with aiPB2-12 and aiNP-4 can significantly inhibit viral replication in MDCK cells.

[0106] 2. Detection of NP protein in cell supernatant

[0107] At 48 h after viral infection, the expression of influenza virus NP protein in cell supernatant was analyzed by Western blot (see Figure 8). aiRNA showed a certain inhibitory effect on the synthesis of influenza virus NP protein in cell supernatant. Compared with the control group, aiPB2-12 targeting NP protein in the supernatant of A / Guangdong-Maonan / SWL1536 / 2019(H1N1), A / Hong Kong / 2671 / 2019(H3N2), A / Guangzhou / 39715 / 2014(H5N6), A / Anhui / 1 / 2013(H7N9), and A / Guangxi / NN10.19T-NGS / 2018(H9N2) cells showed inhibitory effects of 38%, 85%, 64%, 41%, and 63%, respectively. aiPB1-4 targeting A / Hong The inhibitory effects of aiPA-2 on NP protein in the supernatant of Kong / 2671 / 2019(H3N2), A / Guangzhou / 39715 / 2014(H5N6), A / Anhui / 1 / 2013(H7N9), and A / Guangxi / NN10.19T-NGS / 2018(H9N2) cells were 18%, 40%, 52%, and 34%, respectively; aiPA-2 targeting A / Guangdong-Maonan / SWL1536 / 2019(H1N1) and A / Hong The inhibitory effects of aiNP-4 on NP protein in the supernatant of Kong / 2671 / 2019(H3N2), A / Guangzhou / 39715 / 2014(H5N6), A / Anhui / 1 / 2013(H7N9), and A / Guangxi / NN10.19T-NGS / 2018(H9N2) cells were 52%, 83%, 52%, 39%, and 35%, respectively; aiNP-4 targeting A / Guangdong-Maonan / SWL1536 / 2019(H1N1) and A / Hong The inhibitory effects of Kong / 2671 / 2019(H3N2), A / Guangzhou / 39715 / 2014(H5N6), A / Anhui / 1 / 2013(H7N9), and A / Guangxi / NN10.19T-NGS / 2018(H9N2) cell supernatants on NP protein were 58%, 76%, 45%, 32%, and 30%, respectively.

[0108] 3. Detection of intracellular NP protein

[0109] At 48 h after viral infection, the expression of influenza virus NP protein in cells was analyzed by Western blot (see Figure 9). siRNA showed a certain inhibitory effect on the synthesis of influenza virus NP protein in cells. Compared with the control group, aiPB2-12 targeting NP protein in the supernatant of A / Guangdong-Maonan / SWL1536 / 2019(H1N1), A / Guangzhou / 39715 / 2014(H5N6), A / Anhui / 1 / 2013(H7N9), and A / Guangxi / NN10.19T-NGS / 2018(H9N2) cells showed inhibitory effects of 74%, 90%, 86%, and 40%, respectively. aiPB1-4 targeting A / Hong The inhibitory effects of aiPA-2 on NP protein in the supernatant of cells from Kong / 2671 / 2019(H3N2), A / Guangzhou / 39715 / 2014(H5N6), A / Anhui / 1 / 2013(H7N9), and A / Guangxi / NN10.19T-NGS / 2018(H9N2) were 69%, 84%, 76%, and 73%, respectively; The inhibitory effects of aiNP-4 on NP protein in the supernatant of 15 / 2014(H5N6), A / Anhui / 1 / 2013(H7N9), and A / Guangxi / NN10.19T-NGS / 2018(H9N2) cells were 47%, 66%, 77%, and 73%, respectively; the inhibitory effects of aiNP-4 on NP protein in the supernatant of A / Anhui / 1 / 2013(H7N9) and A / Guangxi / NN10.19T-NGS / 2018(H9N2) cells were 78% and 41%, respectively.

[0110] This invention designs siRNAs targeting the polymerase protein and nucleoprotein genes of influenza A virus subtypes H1, H3, H5, H7, and H9. Cellular experiments validated that siPB2-12 and siNP-4 exhibit antiviral activity against different influenza virus subtypes. For the potentially active siRNAs, this study designed their corresponding aiRNAs, and cellular experiments validated that aiPB2-12 and aiNP-4 also possess antiviral activity against different influenza virus subtypes. These results indicate that siPB2-12, siNP-4, and their corresponding aiRNAs possess potential broad-spectrum and specificity.

[0111] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. The use of a double-stranded RNA molecule, or a pharmaceutically acceptable salt thereof, in the preparation of an influenza virus inhibitor, wherein the double-stranded RNA molecule is aiPB2-12 and / or aiNP-4, wherein one strand sequence of aiPB2-12 is SEQ ID No. 3 in the sequence listing and the other strand sequence is SEQ ID No. 4 in the sequence listing, wherein one strand sequence of aiNP-4 is SEQ ID No. 7 in the sequence listing and the other strand sequence is SEQ ID No. 8 in the sequence listing, and wherein the influenza virus is H1N1 and / or H5N6 virus.

2. The use of biological materials containing the double-stranded RNA molecule as described in claim 1 in the preparation of influenza virus inhibitors, wherein the biological material is a DNA molecule capable of producing the double-stranded RNA molecule, a recombinant vector containing the DNA molecule, or a recombinant microorganism containing the DNA molecule, and the influenza virus is H1N1 and / or H5N6 virus.

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