Application of mun-miR-215-5p in preparation of medicine for regulating parrot beak and feather disease virus replication

Abstract

The invention relates to the field of molecular markers, and particularly provides an application of mun-miR-215-5p in preparation of a medicine for regulating virus replication of beak and feather diseases, and the nucleotide sequence of the mun-miR-215-5p is shown as SEQ ID NO. 1. The invention also relates to a preparation method of the mun-miR-215-5p in preparation of a medicine for regulating virus replication of beak and feather diseases. The invention provides a new target for researching drugs for antagonizing virus replication of beak and feather diseases.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of antiviral drugs, and particularly relates to application of mun-miR-215-5p in preparation of a drug for regulating Psittacine Beak and Feather Disease Virus (PBFDV) replication. BACKGROUND

[0002] Psittacine Beak and Feather Disease (PBFD) is a fatal infectious disease caused by a single-stranded circular DNA virus (Psittacine Beak and Feather Disease Virus, PBFDV) in the Circoviridae family, and is mainly characterized by regressive feather changes, beak deformity and immunosuppression. The virus is about 14 nm in diameter, and the genome size is about 1.7-2.0 kb, which encodes two main proteins, i.e. capsid protein (Cap, C1 gene) and replication-related protein. PBFDV mainly spreads through dander, feces and vertical transmission, has strong resistance to acidic environment, chloroform and high temperature, has strong survival ability in the environment, and has poor effect of conventional disinfection. The disease mainly infects birds in the Psittacine order, and young birds are more susceptible. PBFD has become a major epidemic disease threatening the health of global parrot populations, especially posing a serious threat to endangered parrot species. The existing prevention and control measures mainly rely on isolation and quarantine and environmental disinfection, and there is no specific treatment drug and commercial vaccine, so it is urgent to develop new antiviral strategies.

[0003] MicroRNA (miRNA) is a small non-coding RNA of about 20-25 nucleotides in length in vivo, which negatively regulates the expression of genes at the post-transcriptional level by complementary pairing with target mRNA, mainly by inhibiting mRNA translation or inducing mRNA degradation. MiRNA is involved in the regulation of various biological processes such as animal development, cell proliferation and death, and immune response. In recent years, more and more studies have shown that miRNA is an indispensable regulator in the complex network of host innate / acquired immunity and host-pathogen interaction, and plays an important role in host-virus interaction. Studies have shown that certain host miRNAs can directly or indirectly affect the viral replication cycle, inhibit viral replication by regulating host factors or directly targeting viral genomes, which provides a new idea and new target for the development of antiviral drugs. However, there is currently no research on host miRNAs regulating PBFDV replication, and the related antiviral mechanisms are not clear.

[0004] This invention is the first to discover the regulatory role of mun-miR-215-5p in PBFDV replication. Experiments confirmed that upregulating mun-miR-215-5p expression in primary parrot cell alveolar macrophages significantly inhibited PBFDV replication, while downregulating its expression promoted viral replication. This discovery not only provides a new perspective on understanding the host-PBFDV interaction mechanism but also offers a novel target for developing miRNA-based anti-PBFDV drugs. Compared to traditional antiviral strategies, miRNA-based therapies have advantages such as strong targeting and fewer side effects, and are expected to become a novel and effective means of preventing and treating PBFDV. Summary of the Invention

[0005] The present invention addresses the above-mentioned technical problems by providing a composition for inhibiting the replication of beak feather virus, characterized in that the composition comprises a nucleotide sequence as shown in SEQ ID NO.1.

[0006] On the other hand, the present invention provides the use of a drug with increased mun-miR-215-5p content in the preparation of a drug for inhibiting the replication of beak feather virus, characterized in that the sequence of said mun-miR-215-5p is as shown in SEQ ID NO.1.

[0007] On the other hand, the present invention provides the use of a drug that increases the content of mun-miR-215-5p in the preparation of a drug for treating parrot beak and feather disease, characterized in that the sequence of mun-miR-215-5p is as shown in SEQ ID NO.1.

[0008] Preferably, the aforementioned drug for increasing mun-miR-215-5p content is a nucleic acid, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0009] On the other hand, the present invention provides the use of the mun-miR-215-5p inhibitor in the preparation of a drug that promotes the replication of beak feather virus, characterized in that the sequence of the mun-miR-215-5p is as shown in SEQ ID NO.1.

[0010] Preferably, the mun-miR-215-5p inhibitor is a nucleic acid, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0011] On the other hand, the present invention provides a non-disease treatment method for inhibiting the replication of beak feather virus, characterized in that the method is achieved by increasing the expression of mun-miR-215-5p in cells, the sequence of which is shown in SEQ ID NO.1.

[0012] On the other hand, the present invention provides a non-disease treatment method for promoting the replication of beak feather disease virus, characterized in that the method is achieved by reducing the expression of mun-miR-215-5p in cells, the sequence of which is shown in SEQ ID NO.1.

[0013] Preferably, the reduction of mun-miR-215-5p expression in cells is achieved through a mun-miR-215-5p inhibitor.

[0014] Preferably, the mun-miR-215-5p inhibitor is a nucleic acid, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0015] On the other hand, the present invention provides a positive control method for evaluating the effectiveness of a method for inhibiting the replication of beak feather virus, characterized in that the positive control method inhibits the replication of beak feather virus by increasing the expression of mun-miR-215-5p in cells.

[0016] On the other hand, the present invention provides a positive control method for evaluating the effectiveness of a method for promoting the replication of beak feather virus, characterized in that the positive control method promotes the replication of beak feather virus by reducing the expression of mun-miR-215-5p in cells.

[0017] Preferably, the reduction of mun-miR-215-5p expression in cells is achieved through a mun-miR-215-5p inhibitor.

[0018] Preferably, the mun-miR-215-5p inhibitor is a nucleic acid, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0019] This invention provides the application of mun-miR-215-5p in the preparation of drugs that regulate beak feather virus replication. Experimental results show that upregulating mun-miR-215-5p expression in parrot alveolar macrophages inhibits beak feather virus replication, while downregulating its expression promotes it. This invention provides a new target for studying drugs antagonizing beak feather virus replication, lays the foundation for developing new antiviral strategies, and provides candidate miRNAs for a deeper understanding of the host's antagonistic mechanism against beak feather virus replication after infection.

[0020] Compared with existing technologies, this invention provides a new target for the preparation of drugs that antagonize the replication of beak feather virus, and lays the foundation for the development of new antiviral strategies. Transfection of mun-miR-215-5p mimic at a final concentration of 50 nM inhibited the virus by more than 50%. Attached Figure Description

[0021] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0022] Figure 1 Parrot alveolar macrophages were inoculated with beak feather disease virus at MOI=1. Total cellular miRNA was extracted 24 hours later. Real-time quantitative PCR results were obtained. ** indicates P < 0.01.

[0023] Figure 2 Parrot alveolar macrophages were transfected with a mimic of mun-miR-215-5p. Total miRNA was extracted from the cells 24 hours later. Real-time quantitative PCR results were obtained. ** indicates P < 0.01.

[0024] Figure 3 Parrot alveolar macrophages were transfected with an inhibitor of mun-miR-215-5p. Total miRNA was extracted from the cells 24 hours later. Real-time quantitative PCR results were obtained. * indicates P < 0.05.

[0025] Figure 4 Parrot alveolar macrophages were transfected with a mimic of mun-miR-215-5p. After 24 hours, they were inoculated with beak feather disease virus at MOI=1. Viral DNA was extracted 24 hours after infection. Real-time quantitative PCR results were obtained. * indicates P < 0.05.

[0026] Figure 5 Parrot alveolar macrophages were transfected with an inhibitor of mun-miR-215-5p. After 24 hours, they were inoculated with beak feather disease virus at MOI=1. Total DNA was extracted 24 hours after infection. Real-time quantitative PCR results were obtained. ** indicates P < 0.01. Detailed Implementation

[0027] This invention provides the use of mun-miR-215-5p in the preparation of a drug that regulates the replication of beak feather virus, wherein the nucleotide sequence of mun-miR-215-5p is 5'-AUGACCUAUGAAUUGACAGACU-3' (SEQ ID NO.1).

[0028] This invention also provides the application of a substance that upregulates mun-miR-215-5p expression in the preparation of a drug antagonizing beak feather virus replication, the nucleotide sequence of which is shown in SEQ ID NO.1. In a specific embodiment of this invention, 24 hours after infection with the virus strain, the expression level of mun-miR-215-5p in parrot alveolar macrophages decreased. Upregulating the expression level of mun-miR-215-5p in parrot alveolar macrophages (SEQ ID NO.1) can inhibit beak feather virus replication, while downregulating the expression level of this miRNA (using a mun-miR-215-5p inhibitor, SEQ ID NO.2, 5'-AGUCUGUCAAUUCAUAGGUCAU-3') can promote beak feather virus replication. This invention provides a new target for studying drugs antagonizing beak feather virus replication and also provides candidate miRNAs for a deeper understanding of the host antiviral signaling pathway after beak feather virus infection.

[0029] The present invention is further illustrated below with reference to specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified in the following examples, all experimental methods can be performed according to conventional methods, such as those described in J. Sambrook et al.'s *Molecular Cloning: A Laboratory Manual*, F. Osber et al.'s *A Concise Laboratory Manual of Molecular Biology*, or according to the manufacturer's instructions for use of the product.

[0030] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0031] Example 1: Effect of PBFDV infection on mun-miR-215-5p expression in parrot alveolar macrophages (1) Isolation of alveolar macrophages from parrots: Healthy budgies (Melopsittacus undulatus) at 20 weeks of age were selected. After anesthesia and euthanasia, the lungs were aseptically isolated and irrigated with sterile PBS buffer containing 1% penicillin and antibiotics. The collected irrigating fluid was centrifuged at 500 g for 10 min, the supernatant was discarded, and the cells were resuspended in 10 mL of PBS buffer. After washing twice, the cell pellet was collected and resuspended in RPMI-1640 complete medium containing 10% FBS. After counting, the cell concentration was adjusted to 1.5 x 10⁻⁶ cells / mL. 6 10 cells / mL, 1 mL per well, seeded into a 12-well plate and cultured at 37°C. The adherent cells are parrot alveolar macrophages.

[0032] (2) Virus inoculation: PBFDV was inoculated into parrot alveolar macrophages at MOI=1, and a control without virus inoculation was set up. RPMI-1640 medium containing 2% FBS was added to 1 mL and cultured in an incubator at 37 ℃.

[0033] (3) Validation of differential expression of mun-miR-215-5p: Total RNA was extracted from cells treated with different methods using the MolPure® Cell / Tissue Total RNA Kit from Yisheng Biotechnology (Shanghai) Co., Ltd. Reverse transcription was performed using the PrimeScript™ RT reagent Kit with gDNA Eraser from TAKARA (containing a neck loop primer and a random primer; the neck loop primer sequence is 5'-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGAGTCTGTC-3' (SEQ ID NO. 3)) to obtain cDNA suitable for qPCR. qPCR was then performed using the TB Green® Fast qPCR Mix kit from TAKARA. The qPCR reaction was performed using a Bio-Rad CFX96 real-time PCR instrument. The upstream primer sequence for mun-miR-215-5p was 5'-GGGATGACCTATGAATTGA-3' (SEQ ID NO.4), and the downstream primer was 5'-TGGTGTCGTGGAGTCG-3' (SEQ ID NO.5). The upstream primer for the internal control was 5'-CTCGCTTCGGCAGCACA-3' (SEQ ID NO.6), and the downstream primer was 5'-AACGCTTCACGAATTTGCGT-3' (SEQ ID NO.7). The qPCR reaction program was: 95℃, 5 min; 35 cycles (94℃, 15 s; 60℃, 30 s). The T-test was used in SPSS 22.0 software to test the significance of differences in mun-miR-215-5p expression among different treatment groups. Figure 1 The results showed that the level of mun-miR-215-5p was significantly reduced (P<0.01) after PBFDV infection of parrot alveolar macrophages.

[0034] Example 2: Regulation of mun-miR-215-5p expression level in parrot alveolar macrophages (1) Upregulation of mun-miR-215-5p expression level in parrot alveolar macrophages: 4 μL of Lipofectamine transfection reagent (Invitrogen) and 100 μL of OPTI-MEM culture medium (Invitrogen) were mixed and incubated at room temperature for 10 min. 50 pmol of mun-miR-215-5p mimic (5'-AUGACCUAUGAAUUGACAGACU-3') was diluted with 100 μL of OPTI-MEM culture medium, and the two were mixed and incubated at room temperature for 10 min. The diluted mun-miR-215-5p mimic and transfection reagent were mixed and incubated at room temperature for 10 min to form a transfection complex. Parrot alveolar macrophages pre-seeded in 12-well plates were rinsed with OPTI-MEM medium. A mixture of mimicry and liposomes was added to each well, followed by the addition of 790 μL of 10% FBS RPMI-1640 complete medium. The cells were gently mixed and incubated at 37 °C with 5% CO2. Cells were harvested after 24 hours, total RNA was extracted, reverse transcribed, and the expression level of mun-miR-215-5p was detected by real-time quantitative PCR. Figure 2 The results showed that transfection with the mun-miR-215-5p mimic significantly upregulated the expression of mun-miR-215-5p, increasing the expression level by 7.5-fold (P < 0.01).

[0035] (2) Downregulation of mun-miR-215-5p expression level in parrot alveolar macrophages: 4 μL of Lipofectamine transfection reagent (Invitrogen) and 100 μL of OPTI-MEM culture medium (Invitrogen) were mixed and incubated at room temperature for 10 min. 50 pmol of mun-miR-215-5p inhibitor (5'-AGUCUGUCAAUUCAUAGGUCAU-3') was diluted with 100 μL of OPTI-MEM culture medium, and the two were mixed and incubated at room temperature for 10 min. The diluted mun-miR-215-5p inhibitor and transfection reagent were mixed and incubated at room temperature for 10 min to form a transfection complex. Parrot alveolar macrophages pre-seeded in 12-well plates were washed with OPTI-MEM medium. A mixture of inhibitor and liposomes was added to each well, followed by 790 μL of 10% FBS RPMI-1640 complete medium. The cells were gently mixed and incubated at 37 ℃ with 5% CO2. Cells were harvested after 24 hours, total RNA was extracted, reverse transcribed, and the expression level of mun-miR-215-5p was detected by real-time quantitative PCR. Figure 3The results showed that after transfection with the mun-miR-215-5p inhibitor, the expression of mun-miR-215-5p was significantly downregulated (P<0.05).

[0036] Example 3: Verifying the effect of mun-miR-215-5p levels in parrot alveolar macrophages on beak feather disease virus replication. (1) Effect of mun-miR-215-5p mimic on the replication of beak feather virus: Parrot alveolar macrophages were transfected with mun-miR-215-5p mimic at a final concentration of 50 nM for 24 hours. Beak feather virus was then inoculated into the cells at an MOI of 1. Virus was harvested after 24 hours, the supernatant was discarded, and cells were collected. Viral DNA was extracted using the EasyPure® Simple Viral DNA / RNA Kit (Beijing TransGold). The copy number of beak feather virus was determined by absolute quantitative PCR. The upstream primer sequence of the beak feather virus C1 gene was 5'-TTGGGTCCTCCTTGTAGTGGGATC-3', and the downstream primer sequence was 5'-CAGACGCCGTTTCTCAACCAATAG-3'. Figure 4 The results showed that upregulation of mun-miR-215-5p expression decreased the replication level of beak feather virus (P < 0.05). This suggests that mun-miR-215-5p has the function of inhibiting beak feather virus replication.

[0037] (2) Effect of mun-miR-215-5p inhibitor on the replication of beak feather virus: Parrot alveolar macrophages were transfected with mun-miR-215-5p inhibitor at a final concentration of 50 nM for 24 hours. Beak feather virus was then inoculated into the cells at an MOI of 1. The virus was harvested after 24 hours, the supernatant was discarded, and the cells were collected. Viral DNA was extracted, and the copy number of beak feather virus was determined by absolute quantitative PCR. Figure 5 The results showed that downregulation of mun-miR-215-5p expression increased the replication level of beak feather disease virus (P < 0.01). This suggests that mun-miR-215-5p inhibitors have the function of promoting viral replication.

[0038] Based on the disclosure and teachings of the foregoing specification, those skilled in the art can make appropriate changes and modifications to the above embodiments. Therefore, the present invention is not limited to the specific embodiments disclosed and described above, and some modifications and changes to the present invention should also fall within the protection scope of the claims of the present invention. Furthermore, although some specific terms are used in this specification, these terms are only for convenience of explanation and do not constitute any limitation on the present invention.

Claims

1. A composition for inhibiting the replication of a beak disease virus, characterized by, The composition comprises a nucleotide sequence as shown in SEQ ID NO.

1.

2. The use of a drug for increasing the content of mun-miR-215-5p in the preparation of a drug for inhibiting the replication of the Avian Leukosis Virus, characterized in that, The sequence of the mun-miR-215-5p is shown in SEQ ID NO.

1.

3. The use of a drug for increasing the content of mun-miR-215-5p in the preparation of a drug for treating beak and feather disease in parrots, characterized in that, The sequence of the mun-miR-215-5p is shown in SEQ ID NO.

1.

4. Use of a mun-miR-215-5p inhibitor in the preparation of a medicament for promoting the replication of a beak and feather disease virus, characterized in that, The sequence of the mun-miR-215-5p is shown in SEQ ID NO.

1.

5. A non-disease treatment method of inhibiting the replication of a Beak and Feather Disease virus, characterized in that, The method is achieved by increasing the expression of mun-miR-215-5p in the cell, and the sequence of the mun-miR-215-5p is shown in SEQ ID NO.

1.

6. A non-disease treatment method for promoting replication of Avian Leukosis Virus, characterized by, The method is achieved by reducing the expression of mun-miR-215-5p in the cell, and the sequence of the mun-miR-215-5p is shown in SEQ ID NO.

1.

7. The method of claim 6, wherein, The reduction of the expression of mun-miR-215-5p in the cell is achieved by a mun-miR-215-5p inhibitor.

8. A positive control method for evaluating the effect of inhibiting the replication of a Beak and Feather Disease virus, characterized by, The positive control method is to inhibit the replication of the beak feather disease virus by increasing the expression of mun-miR-215-5p in the cell, and the sequence of the mun-miR-215-5p is shown in SEQ ID NO.

1.

9. A positive control method for evaluating the effect of promoting the replication of beak feather disease virus, characterized by, The positive control method is to promote the replication of the beak feather disease virus by reducing the expression of mun-miR-215-5p in the cell, and the sequence of the mun-miR-215-5p is shown in SEQ ID NO. 1.

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