Triphosphorylation modified miRNA and application thereof in virus resistance

Abstract

The invention relates to the technical field of immunotherapy, in particular to triphosphorylation modified miRNA (micro Ribonucleic Acid) and application thereof in virus resistance. The miRNA molecule carrying 5 '-PPP modification is obtained through in-vitro transcription synthesis of T7 RNA polymerase by using a specific template, the miRNA can improve the expression quantity of RIG-I and IFN-beta and actively activate an RIG-I pathway inhibited by PRRSV so as to reconstruct an antiviral state in a cell, and also can specifically target and silence a PRRSV genome and reduce the risk of virus mutation escape, so that the miRNA molecule can be used for preparing an anti-viral drug. Therefore, the replication of the PRRSV in the cells is more efficiently inhibited.

Description

Technical Field

[0001] This invention relates to the field of immunotherapy technology, and in particular to triphosphorylated miRNAs and their application in antiviral therapy. Background Technology

[0002] Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV), resulting in reproductive failure in sows and respiratory disorders in pigs of all ages, causing enormous economic losses to the global pig industry. PRRSV is a single-stranded positive-sense RNA virus belonging to the arteritis virus family. The PRRSV genome is approximately 15 kb in length, containing 11 open reading frames, a 5' untranslated region (UTR), and a 3' UTR. These genes encode various structural proteins (such as GP5 and M proteins) and non-structural proteins (such as nsp2), some of which are closely related to viral immune evasion and pathogenicity. However, due to the high frequency of mutations in the viral genome, genetic recombination among different lineages / sublineages, and the immunosuppression caused by PRRSV, there are currently no effective methods to prevent PRRSV.

[0003] Plant-derived small RNAs (sRNAs) are a class of 20-24 nt non-coding RNAs that mediate the degradation or translational repression of target genes by binding complementary to the 3' untranslated region of target mRNAs. Many sRNAs derived from natural plants have shown great antiviral potential. MiR2911, a miRNA derived from honeysuckle, can directly target the genomes of viruses such as influenza A virus, enterovirus 71, and SARS-CoV-2 to inhibit their replication. Recent studies have shown that miR2911 can also specifically target the PRRSV genome, thereby inhibiting PRRSV replication in vitro. However, this strategy still has certain limitations. The high mutation rate of RNA viruses allows them to rapidly evolve and weaken the miRNA-mediated repressive effect; therefore, a single miRNA may not be able to achieve durable and effective gene silencing. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides triphosphorylated miRNAs and their applications in antiviral therapy. The triphosphorylated miRNAs provided by this invention can both activate the host's innate immunity and specifically target and silence the PRRSV genome, thereby more efficiently inhibiting PRRSV replication in cells.

[0005] To achieve the above objectives, the present invention provides the following technical solution: This invention provides triphosphorylated modified miRNAs, wherein the miRNAs include 5'PPP miR181c and / or 5'PPPBZL-sRNA-20; the 5'PPP miR181c is synthesized in vitro by T7 RNA polymerase using a nucleotide sequence as a template, as shown in SEQ ID NO.3 or SEQ ID NO.4; the 5'PPP BZL-sRNA-20 is synthesized in vitro by T7 RNA polymerase using a nucleotide sequence as a template, as shown in SEQ ID NO.5 or SEQ ID NO.6.

[0006] Preferably, the kit used for the in vitro transcription synthesis includes the HiScribe T7 Quick High YieldRNA Synthesis Kit.

[0007] This invention provides the application of the miRNA described in the above technical solution in the preparation of antiviral products.

[0008] Preferably, the virus includes porcine reproductive and respiratory syndrome virus (PRRSV).

[0009] Preferably, the antiviral treatment includes activating the host's innate immunity and / or inhibiting viral replication.

[0010] Preferably, the activation of the host's innate immunity includes increasing the expression levels of RIG-I and / or IFN-β.

[0011] Preferably, the inhibition of viral replication includes silencing the viral genome and / or reducing viral titer.

[0012] Preferably, the product includes a drug or a feed additive.

[0013] This invention provides an antiviral drug, the active ingredient of which includes the miRNA described in the above technical solution.

[0014] This invention provides a feed additive comprising the miRNA described in the above-mentioned technical solution.

[0015] Beneficial effects: This invention provides a triphosphorylated miRNA comprising 5'PPP miR181c and / or 5'PPP BZL-sRNA-20. The 5'PPP miR181c is synthesized in vitro via T7 RNA polymerase using a nucleotide sequence as a template, as shown in SEQ ID NO.3 or SEQ ID NO.4. The 5'PPP BZL-sRNA-20 is synthesized in vitro via T7 RNA polymerase using a nucleotide sequence as a template, as shown in SEQ ID NO.5 or SEQ ID NO.6. This invention, through in vitro transcription with T7 RNA polymerase using a specific template, yields a miRNA molecule carrying 5'-PPP modification. This miRNA not only increases the expression levels of RIG-I and IFN-β, actively activating the RIG-I pathway suppressed by PRRSV, thereby restoring the antiviral state within the cell, but also specifically targets and silences the PRRSV genome, reducing the risk of viral mutation escape, thus more efficiently inhibiting PRRSV replication in cells. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0017] Figure 1 The results show the experimental findings of in vitro transcription of 5'PPP miRNA to activate the RIG-I pathway; where A is a schematic diagram of in vitro transcription to generate 5'PPP miRNA; B is... RIG-I Relative gene expression level; C is... IFN-β Relative expression level of the gene; D is the concentration of IFN-β protein in the supernatant; Figure 2 The results show the experimental findings of 5'PPP BZL-sRNA-20 strongly inhibiting viral replication in vitro; where A represents the ORF7 gene expression level; B represents the viral genome copy number in the supernatant; C represents the viral titer in the supernatant; D represents the Western blot results; E represents the immunofluorescence detection results; and F represents the results in cells. ORF7 RT-qPCR results of the gene; G represents the Western blot result of ORF7 protein in cells; Figure 3 The experimental results show that 5'PPP BZL-sRNA-20 can directly target the viral genome; where A is the PRRSV genome sequence targeted by BZL-sRNA-20, and different shades of gray represent gene differences; B is the PRRSV phylogenetic tree; and C is the detection results of dual-luciferase reporter protein. Figure 4The experimental results show that miR181c and 5'PPP miRNA can inhibit viral replication in PAM cells; where A is... ORF7 Gene expression level; B represents the viral titer in the supernatant. Detailed Implementation

[0018] This invention provides triphosphorylated miRNAs, said miRNAs comprising 5'PPP miR181c and / or 5'PPPBZL-sRNA-20; the 5'PPP miR181c is synthesized in vitro via T7 RNA polymerase using a nucleotide sequence as a template, as shown in SEQ ID NO.3 or SEQ ID NO.4; the 5'PPP BZL-sRNA-20 is synthesized in vitro via T7 RNA polymerase using a nucleotide sequence as a template, as shown in SEQ ID NO.5 or SEQ ID NO.6. As one embodiment, the kit used for the in vitro transcription synthesis includes the HiScribe T7 Quick High Yield RNASynthesis Kit.

[0019] This invention synthesizes miRNA molecules carrying 5'-PPP modification (5'PPP miR181c or 5'PPP BZL-sRNA-20) through in vitro transcription using T7 RNA polymerase with a specific template. Transfection of these miRNAs into cells revealed that the 5'-PPP miRNA significantly increased RIG and IFN-β levels, while the activation effect disappeared upon removal of phosphorylation modification, confirming that 5'-PPP is a key factor in activating RIG-I. Subsequent verification of its antiviral effect in Marc145 cells showed that only 5'PPP BZL-sRNA-20 strongly inhibited viral replication, significantly reduced viral ORF7 transcription and protein levels, and substantially decreased viral titer. Dual-luciferase reporter assays further demonstrated that 5'PPP BZL-sRNA-20 can directly target the viral genome, while unmodified BZL-sRNA-20 did not have this effect, indicating that its targeting function is dependent on 5'PPP modification.

[0020] This invention was validated in the natural host cell, PAM cells. Unlike Marc145 cells where only 5'PPPBZL-sRNA-20 showed significant inhibitory effects, in PAM cells, all 5'PPP-modified miRNAs inhibited viral replication. Figure 4 (A and B). Among them, 5'PPP BZL-sRNA-20 still showed the strongest inhibitory activity, consistent with the previous results. In PAM, an immune-active cell, activation of innate immunity synergistically enhanced the miRNA-based antiviral strategy, thereby more effectively inhibiting PRRSV infection.

[0021] The miRNA provided by this invention is a novel anti-PRRSV miRNA based on activating the host's innate immunity. This miRNA can not only increase the expression levels of RIG-I and IFN-β and actively activate the RIG-I pathway that is inhibited by PRRSV, thereby rebuilding the antiviral state in the cell, but also specifically target and silence the PRRSV genome, reducing the risk of viral mutation escape, thereby inhibiting PRRSV replication in the cell more efficiently.

[0022] Based on the above advantages, the present invention provides the application of the miRNA described in the above technical solution in the preparation of antiviral products.

[0023] In one embodiment, the antiviral treatment includes activating the host's innate immunity and / or inhibiting viral replication. In one embodiment, activating the host's innate immunity includes increasing the expression levels of RIG-I and / or IFN-β. In one embodiment, inhibiting viral replication includes silencing the viral genome and / or reducing viral titers. In one embodiment, the virus includes porcine reproductive and respiratory syndrome virus (PRRSV).

[0024] In one implementation, the product includes a drug or feed additive.

[0025] Based on the above advantages, the present invention provides an antiviral drug, the active ingredient of which includes the miRNA described in the above technical solution.

[0026] Based on the above advantages, the present invention provides a feed additive comprising the miRNA described in the above technical solution.

[0027] To further illustrate the present invention, the triphosphorylated miRNAs provided by the present invention and their application in antiviral therapy are described in detail below with reference to embodiments and accompanying drawings, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0028] Example 1. Cells and Viruses Marc145 cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. PAM cells were obtained by lung lavage of 6-week-old pathogen-free (SPF) pigs and maintained in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin. The NADC30-like strain, the PRRSV strain isolated and identified in this invention, was propagated and titrated in Marc145 cells.

[0029] 2. Preparation of different miRNAs 1) Using the HiScribe T7 Quick High Yield RNA Synthesis Kit (NewEngland Biolabs, NEB, E2050S), miRNAs with 5' triphosphates (5'PPP miRNC, 5'PPP miR181c, and 5'PPP BZL-sRNA-20) were produced via in vitro transcription, including the following steps: The oligonucleotides required for in vitro transcription were synthesized by Beijing Tianyi Huiyuan Company, which contain a T7 RNase promoter region (5'-TAATACGACTCACTATAG-3', SEQ ID NO.7) and miRNA sequence, as detailed in Table 1.

[0030] Table 1. Oligonucleotides required for in vitro transcription of miRNAs with 5' triphosphates.

[0031] DNA templates were constructed by annealing the single-stranded oligonucleotides listed in Table 1. The oligonucleotides were incubated with the annealing solution at 90°C for 5 min, then cooled to 45°C at a rate of -1°C / min. The annealing reaction mixture consisted of 3 μL of 10× annealing solution, 21 μL of RNase-free H₂O, 3 μL of sense oligonucleotides, and 3 μL of antisense oligonucleotides. The 10× annealing solution was composed of 250 mM Tris-HCl and 250 mM NaCl, with a pH of 7.4. Transcription was performed overnight at 37°C according to the instructions of the HiScribe T7 Quick High Yield RNA Synthesis Kit, and excess DNA template was digested with DNase I. RNA was purified using MonarchRNA Cleanup (NEB, T2050L) and its concentration was measured using a NanoDrop™ 2000 (Thermo Fisher Scientific). The obtained 5'PPP miRNA was electrophoresed on a 15% TBE-urea gel, and the target RNA was purified using small RNA PAGE Recovery (Zymo Research, R1070).

[0032] 2) Following the in vitro transcription method of step 1), miRNAs with 5' triphosphate were produced. The 5' triphosphate was then removed using bovine intestinal alkaline phosphatase CIP (NEB, M0525V) to obtain three post-transcriptionally de-5' triphosphate miRNAs (CIPmiRNC, CIP miR181c, and CIP BZL-sRNA-20). 500 ng of 5' PPP miRNA was incubated with 1 μL of CIP at 37°C for 40 min to ensure complete reaction.

[0033] 3) Jiangsu Saisofe Biotechnology Co., Ltd. was commissioned to synthesize unmodified miRNA (Table 2).

[0034] Table 2 Unmodified miRNAs

[0035] 3. miRNA transfection and viral infection According to the manufacturer's instructions, miRNA was transfected into Marc145 or PAM cells at a final concentration of 20 nM using the TransIT-X2 Dynamic Delivery System (Mirus Bio, MIR600), except for dose-dependent experiments. For viral infection, the cells were infected with a NADC30-like strain at 0.1 MOI 24 h after transfection. Cells or supernatant were harvested for the next step of the experiment 24 h after infection.

[0036] 4. RT-qPCR According to the manufacturer's instructions, total RNA was extracted from cells or supernatant using Direct-zol™ RNA Miniprep (Zymo Research, R2050). Subsequently, the RNA was reverse transcribed using TransScript All-in-One First-Strand cDNASynthesis Supermix (Transgen Biotech, AT341), and quantitative qPCR analysis was performed using PerfectStartVisual Green qPCR SuperMix (Transgen Biotech, AQ621). β-actin was used as an internal control protein. The relative gene expression level was calculated using a method. Primers used for qPCR are shown in Table 3.

[0037] Table 3 Primers used for qPCR

[0038] For the detection of PRRSV in the supernatant, refer to the literature [Zhang, A.; Zhao, L.; Li, N.; Duan, H.; Liu, H.; Pu, F.; Zhang, G.; Zhou, EM; Xiao, S. Carbon Monoxide Inhibits Porcine Reproductive and Respiratory Syndrome Virus Replication by the CyclicGMP / Protein Kinase G and NF-kappaB Signaling Pathway]. J Virol 2017, 91 The method disclosed in [doi:10.1128 / JVI.01866-16.] involves serially diluting plasmids containing the ORF7 sequence as standards, plotting a standard curve based on the qPCR results, and then quantifying the viral load in the supernatant using the standard curve.

[0039] 5. ELISA According to the manufacturer's instructions, the IFN-β level in cell culture supernatant was measured using a monkey interferon-β ELISA kit (Finetest, EMK0029). The process included adding the culture supernatant and detection standard solution to an antibody-coated ELISA plate, followed by biotin-labeled antibody solution, HRP-streptavidin, and finally TMB chromogenic substrate and stop solution. OD values ​​were read at 450 nm using an ELISA reader. 450 The numerical values ​​were then converted into concentration values ​​using a standard curve.

[0040] 6. Western blot Collect processed cells and lyse them using RIPA lysis buffer containing fresh protease and phosphatase inhibitors. Transfer samples to a 12% SDS-PAGE gel for electrophoresis. After electrophoresis, transfer the samples to a PVDF membrane and block with rapid blocking buffer (NCM Biotech, P30500). Incubate the membrane overnight at 4°C with the following primary antibodies: β-actin primary antibody (MBL, M177-3) and PRRSV-N primary antibody (GeneTex, GTX129270). Wash the membrane three times with TBST and incubate for 1 hour with the corresponding HRP-labeled secondary antibody. After TBST washing, uniformly drop ECL imaging buffer (Epizyme Biomedical, SQ201) onto the membrane and acquire signals using an imaging system.

[0041] 7. Immunofluorescence The treated cells were fixed with 4% paraformaldehyde for 20 minutes. After washing three times with PBS, the cells were permeabilized with 0.1% Triton X-100 (Yeasen, 20107ES76) diluted in PBS for 20 minutes. After washing with PBS, the cells were blocked by incubating with 10% goat serum for 1 hour. The cells were then incubated overnight at 4°C with PRRSV-N primary antibody (GeneTex, GTX129270). After washing three times with PBS, the cells were incubated with FITC-labeled secondary antibody (Servicebio, GB22403) at room temperature in the dark for 1 hour. The cells were then stained with DAPI (Beyotime, C1006). The stained cells were then observed using a digital confocal imaging system (Thermo Fisher Scientific, EVOS M7000).

[0042] 8. miRNA target gene prediction and conservation analysis RNA-hybrid was used to predict potential BZL-sRNA-20 targets in PRRSV genomic regions with minimum folding energies below -25 kcal / mol. -1 The conservation of target sequences on different downloaded PRRSV strains was compared using MEGA12.0, and a phylogenetic tree was constructed.

[0043] 9. Dual-luciferase reporter The highly conserved BZL-sRNA-20 targeting the PRRSV genomic region was tandemly linked and inserted into the pmirGLO vector (Promega, E1330) using Sac I and Xba I, named pmirGLO-PRRSV. The miRNA and pmirGLO-PRRSV plasmid were co-transfected into Marc145 cells. After 48 hours, the luciferase activities of firefly and Renalis cells were detected using the Duo-Lite Luciferase Assay System (Vazyme, DD1205) according to the manufacturer's instructions.

[0044] 10. Data Analysis Statistical analysis was performed using GraphPad Prism 9.5. Experimental data are expressed as mean ± standard deviation (mean ± SD). N represents the number of biological replicates. p < 0.05 was considered statistically significant. Specifically, it is labeled as (*p < 0.05; **p < 0.01; ***p < 0.001; ***p < 0.0001; ns indicates no statistically significant difference).

[0045] 11. Results and Analysis 1) In vitro transcribed 5'PPP miRNA can activate the RIG-I pathway. To target PRRSV, this invention screened two miRNAs with different mechanisms of action: one is miR-181c, which blocks viral invasion by acting on the host CD163 receptor; the other is BZL-sRNA-20, which targets the host TLR4 and has broad-spectrum antiviral potential. First, these two miRNAs with a 5′ triphosphate (5′PPP) modification were synthesized using in vitro transcription. Figure 1 (A) To verify the specific interaction between 5′PPP modification and the RIG-I receptor, the 5′PPP modification was removed using the phosphatase CIP, converting the 5′ end to a hydroxyl group. Subsequently, unmodified miRNA, 5′PPP miRNA, and CIP-treated miRNA were transfected into Marc145 cells. 48 hours after transfection, cellular RNA was extracted, and the expression of related molecules was detected by qPCR and ELISA. The results are shown in [Figure A]. Figure 1 The results showed that the expression levels of unmodified miR-181c and BZL-sRNA-20 were not significantly different from those of the negative control miRNA (miRNC), indicating that these two miRNAs themselves do not possess the ability to activate innate immunity. Compared with unmodified miRNAs, the 5′PPP miRNA transfection group showed an approximately 20-fold increase in RIG-I expression, an approximately 400-fold increase in IFN-β mRNA levels, and an approximately 200-fold increase in IFN-β protein content in the cell supernatant. Furthermore, in vitro transcribed miRNAs with 5′PPP modification removed via CIP showed that their RIG-I and IFN-β expression levels were restored to similar levels to unmodified miRNAs, with no statistically significant differences. These results indicate that activation of the RIG-I pathway is strictly dependent on 5′PPP modification of miRNAs.

[0046] 2) 5'PPP BZL-sRNA-20 strongly inhibits viral replication in vitro. After confirming the immunostimulatory effect of 5'PPP modification, this invention further evaluated the inhibitory effect of 5'PPP miRNA on PRRSV replication in vitro. First, different miRNAs were transfected into Marc145 cells at a final concentration of 20 nM. Twenty-four hours after transfection, cells were infected with the NADC30-like strain at a MOI of 0.1. Twenty-four hours after infection, cells and culture supernatants were collected, and viral RNA levels were detected by RT-qPCR. The results showed that in the experimental group transfected with 5'PPP BZL-sRNA-20, the viral ORF7 mRNA level decreased by approximately 100-fold (…). Figure 2 In the upper part of the culture medium (A), the viral genome copy number decreased by approximately 55-fold. Figure 2(Middle B). In the remaining treatment groups, except for a slight decrease in viral load in the supernatant of the 5'PPP miR-181c group, there were no significant differences between the other groups and the negative control (miRNC). Viral titer assays further indicated that only 5'PPP BZL-sRNA-20 could cause a significant decrease in viral titer ( Figure 2 (C). Subsequently, the expression of viral N protein was detected by Western blot and immunofluorescence. Consistent with the above results, viral N protein was almost undetectable after transfection with 5'PPP BZL-sRNA-20. Figure 2 (D and E). Furthermore, the 5'PPP BZL-sRNA-20-mediated inhibition was dose-dependent: with increasing transfection concentration, the expression of ORF7 mRNA and its encoded protein in cells gradually decreased (D and E). Figure 2 (F and G). The above results indicate that 5'PPP BZL-sRNA-20 can effectively inhibit PRRSV replication in Marc145 cells.

[0047] 3) 5'PPP BZL-sRNA-20 exerts its antiviral effect by targeting the viral genome. To verify whether 5'PPP BZL-sRNA-20 directly targets the PRRSV genome, this invention first used RNAhybrid software to predict its potential binding sites with the PRRSV genome, identifying a total of 11 candidate targets. To assess the conservation of these targets, we downloaded the genome sequences of several representative PRRSV strains from the NCBI database and constructed a phylogenetic tree to reflect their genetic background diversity. Figure 3 (B) By performing multiple sequence alignment and conservation analysis on all predicted targets, highly evolutionarily conserved target sequences were selected. Figure 3 (A) and excluded two less conserved sites. Subsequently, the highly conserved targets were tandemly linked according to their genomic order and cloned into the pmirGLO dual-luciferase reporter vector. Experimental results showed that 5'PPP BZL-sRNA-20 specifically bound to these conserved targets and significantly inhibited luciferase activity; while unmodified BZL-sRNA-20 and 5'PPP miRNC did not have this effect ( Figure 3 (C). This result confirms that 5'PPP BZL-sRNA-20 can directly target the PRRSV genome, and its targeting function depends on 5'PPP modification.

[0048] 4) miR181c and 5'PPP miRNA can inhibit viral replication in PAM cells. After validating the antiviral activity of 5'PPPBZL-sRNA-20 in the Marc145 cell line, this invention further evaluated its efficacy in its natural host cells—porcine alveolar macrophages. PAM cells are the main target cells for PRRSV infection in vivo, and their inhibitory effect in this model is of great significance for evaluating therapeutic potential. Unlike Marc145 cells, where only 5'PPPBZL-sRNA-20 showed a significant inhibitory effect, in PAM cells, all 5'PPP-modified miRNAs inhibited viral replication (…). Figure 4 (A and B in the original text). Among them, 5'PPP BZL-sRNA-20 still showed the strongest inhibitory activity, consistent with the previous results. Notably, even without 5'PPP modification, miR-181c could still mildly inhibit viral replication by targeting the CD163 receptor on the surface of PAM cells. These results suggest that in PAM, an immune-active cell, activation of innate immunity synergistically enhances miRNA-based antiviral strategies, thereby more effectively inhibiting PRRSV infection.

[0049] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A triphosphorylated miRNA characterized in that, The miRNA comprises 5'PPP miR181c and / or 5'PPP BZL-sRNA-20; the 5'PPP miR181c is synthesized by in vitro transcription of T7 RNA polymerase with the nucleotide sequence as shown in SEQ ID NO. 3 or SEQ ID NO. 4 as a template; and the 5'PPP BZL-sRNA-20 is synthesized by in vitro transcription of T7 RNA polymerase with the nucleotide sequence as shown in SEQ ID NO. 5 or SEQ ID NO. 6 as a template.

2. The miRNA of claim 1, wherein, The kit used for the in vitro transcription synthesis comprises a HiScribe T7 Quick High Yield RNA Synthesis Kit.

3. Use of the miRNA of claim 1 or 2 in the preparation of an antiviral product.

4. Use according to claim 3, characterized in that, The virus comprises a porcine reproductive and respiratory syndrome virus.

5. Use according to claim 3 or 4, characterized in that, The antiviral comprises activating host innate immunity and / or inhibiting viral replication.

6. Use according to claim 5, characterized in that, The activating host innate immunity comprises increasing the expression amount of RIG-I and / or IFN-β.

7. Use according to claim 5, characterized in that, The inhibiting viral replication comprises silencing a viral genome and / or reducing viral titer.

8. Use according to claim 3, characterized in that, The product comprises a medicine or a feed additive.

9. An antiviral medicament, characterized by comprising the compound of claim 1. The active ingredient comprises the miRNA of claim 1 or 2.

10. A feed additive, characterized in that, The product comprises the miRNA of claim 1 or 2.

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