Application of TRIM25 gene as a target point in inhibition of H9N2 influenza virus replication

Abstract

The application belongs to the technical field of genetic engineering, and particularly relates to application of TRIM25 gene as a target point in inhibition of H9N2 influenza virus replication.(1) The application finds that inhibition or silencing of the TRIM25 gene can inhibit replication of the H9N2 influenza virus, and TRIM25 can be used as a target point for screening drugs for inhibiting replication of the H9N2 influenza virus;(2) The application provides an sgRNA specifically targeting the TRIM25 gene, and complete knockout of the TRIM25 gene is realized by combining CRISPR-Cas9 technology, and a monoclonal cell line obtained has a resistance phenotype to the H9N2 influenza virus, thereby providing a new idea for prevention and control of the H9N2 influenza virus;(3) The application finds that a TRIM25 cell line with overexpression can promote replication of the H9N2 influenza virus, and TRIM25 or an expression promoter thereof can be used as a replication or production enhancer of the H9N2 influenza virus.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to the application of the TRIM25 gene as a target in inhibiting the replication of H9N2 influenza virus. Background Technology

[0002] The H9N2 influenza virus belongs to the genus *Influenza Avirus* (IAV) of the family Orthomyxoviridae. It is a segmented, single-stranded, negative-sense RNA virus. The natural hosts of influenza viruses are wild waterfowl and birds. However, the host range of the H9N2 subtype influenza virus is constantly expanding, capable of infecting various mammals, including humans, posing a significant threat to public health. H9N2 is the most common low-pathogenic influenza virus, and while it does not directly cause obvious clinical symptoms, its infection suppresses the immune response in poultry. Furthermore, its prolonged course and susceptibility to secondary infections with other pathogens can cause severe damage and even death in poultry, resulting in significant losses to the poultry farming industry. Currently, vaccination remains one of the main strategies for controlling H9N2 influenza in my country. However, the prevalence of multiple H9N2 viruses with different antigenic compositions in my country, coupled with the fact that vaccine development lags behind the rate of viral mutation, presents a challenge to effective vaccination. Therefore, developing a universal vaccine with broad-spectrum neutralizing activity is of great importance for controlling H9N2. Therefore, further research on the H9N2 influenza virus and revealing more mechanisms by which viral proteins regulate the host's immune response are crucial.

[0003] TRIM25 is an E3 ubiquitin ligase. Ubiquitination is the process by which ubiquitin binds to specific target proteins in the synergistic action of ubiquitin activator E1, ubiquitin coupler E2, and ubiquitin ligase E3. Initial studies found that ubiquitination is involved in protein degradation; further research has revealed that ubiquitination can also mediate protein-protein interactions and cell signal transduction. TRIM25 was the first immunomodulatory TRIM protein identified. It regulates RIG-I activity by controlling K63-type polyubiquitin chain modification of CARDs. This is a non-degradative type of ubiquitination that induces RIG-I oligomerization, which is then recruited to MAVS, thereby triggering the expression of downstream antiviral genes. Existing research shows that TRIM25 expression can inhibit foot-and-mouth disease virus, influenza A virus, infectious bursal disease virus, and rabies virus.

[0004] This invention unexpectedly discovered that, contrary to foot-and-mouth disease virus, influenza A virus, infectious bursal disease virus, and rabies virus, TRIM25 expression can actually promote the replication of H9N2 influenza virus. TRIM25 or its expression promoters can serve as replication or production enhancers for H9N2 influenza virus, and can be used in the production of H9N2 influenza virus or vaccines. Conversely, inhibiting TRIM25 expression can inhibit the replication of H9N2 influenza virus, and can be used as a target for the preparation of drugs that inhibit the replication of H9N2 influenza virus. Based on this, this invention designs a specific sgRNA targeting the TRIM25 gene. The sgRNA specifically targets the TRIM25 gene, and combined with CRISPR-Cas9 technology, TRIM25 gene knockout was achieved. The resulting monoclonal cell line exhibits a resistance phenotype to H9N2 influenza virus and can significantly inhibit the replication of H9N2 influenza virus in cells. This provides research tools and materials for studying the molecular mechanism by which the TRIM25 gene regulates the replication of pathogenic microorganisms in cells, and can also be used for animal breeding against H9N2 influenza virus. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention first discovers that inhibiting or silencing the host TRIM25 gene can suppress the replication of the H9N2 influenza virus, which can be used as a target for preparing drugs that inhibit H9N2 influenza virus replication. Secondly, this invention provides an sgRNA that specifically targets the TRIM25 gene. This sgRNA specifically targets the TRIM25 gene, and by combining it with CRISPR-Cas9 technology, the TRIM25 gene is knocked out. The resulting monoclonal cell line exhibits a resistance phenotype against the H9N2 influenza virus and can significantly inhibit H9N2 influenza virus replication. Furthermore, by targeting the TRIM25 gene, drugs for the prevention or treatment of H9N2 influenza virus infection can be screened, providing a new approach to the control of H9N2 influenza virus. Finally, this invention found that overexpression of TRIM25 can significantly promote the replication of H9N2 influenza virus. Therefore, TRIM25 or its expression promoters can be used as replication or production enhancers of H9N2 influenza virus for the production of H9N2 influenza virus or vaccines. Moreover, cell lines overexpressing TRIM25 can be used as production cell lines for H9N2 influenza virus or vaccines.

[0006] Specifically, it includes the following:

[0007] In a first aspect, the present invention provides an application of the TRIM25 gene / protein as a target in screening drugs for the prevention or treatment of H9N2 influenza virus infection, wherein the drug targets the TRIM25 gene / protein to inhibit or silence the expression of the TRIM25 gene / protein.

[0008] Secondly, this invention provides the use of TRIM25 gene / protein expression inhibitors in the preparation of drugs for the prevention or treatment of H9N2 influenza virus infection.

[0009] Preferably, the inhibitor is an RNA fragment that interferes with the expression of the TRIM25 gene / protein, or an SgRNA that targets the TRIM25 gene / protein, or a small molecule compound that downregulates the expression of the TRIM25 gene / protein.

[0010] Preferably, the inhibitor is an SgRNA that targets the TRIM25 gene / protein.

[0011] Preferably, the inhibitor further includes the mRNA of the Cas9 protein.

[0012] Preferably, the inhibitor is delivered into the animal via a drug delivery vector, carrying sgRNA targeting the TRIM25 gene / protein and mRNA carrying the Cas9 protein to inhibit TRIM25 gene expression.

[0013] Preferably, the drug delivery carrier is a liposome nanoparticle.

[0014] Preferably, the sequence of the SgRNA targeting the TRIM25 gene / protein is as follows:

[0015] TRIM25-sgRNA-F: 5'-GGGAGCCACCCGCCGACGTC-3';

[0016] TRIM25-sgRNA-R: 5'-GACGTCGGCCGGGTGGCTCCC-3'.

[0017] Thirdly, the present invention provides an sgRNA that specifically targets the TRIM25 gene, characterized in that the sequence of the sgRNA is:

[0018] TRIM25-sgRNA-F: 5'-GGGAGCCACCCGCCGACGTC-3';

[0019] TRIM25-sgRNA-R: 5'-GACGTCGGCCGGGTGGCTCCC-3'.

[0020] Fourthly, the present invention provides the application of the sgRNA described in the third aspect above in the preparation of TRIM25 gene knockout cell lines, or in the preparation of drugs for the prevention or treatment of H9N2 influenza virus infection.

[0021] Fifthly, the present invention provides a method for constructing a TRIM25 gene knockout cell line, wherein the method involves using gene targeting technology to cause the TRIM25 gene-encoded protein in the host cell to lose its function.

[0022] Preferably, the method is CRISPR-Cas9 technology.

[0023] Preferably, the method includes the following steps:

[0024] (1) Prepare the sgRNA that specifically targets the TRIM25 gene as described in the third aspect above, add a CACC sticky end to the 5' end of the forward sequence of the sgRNA fragment and add an AAAC sticky end to the 5' end of the reverse sequence to serve as an sgRNA oligonucleotide targeting the TRIM25 gene.

[0025] (2) Insert the double-stranded fragment prepared in step (1) into the multiple cloning site of the Cas9 expression vector to obtain a recombinant vector that simultaneously expresses the Cas9 protein gene and the targeting sgRNA sequence.

[0026] (3) Transfect the host cells with the recombinant vector prepared in step (2), pick a single cell, seed and culture it to obtain the TRIM25 gene knockout cell line.

[0027] In a sixth aspect, the present invention provides a TRIM25 gene knockout cell line constructed according to the method described in the sixth aspect above.

[0028] In a seventh aspect, the present invention provides the application of TRIM25 gene knockout cell lines in detection for non-diagnostic and therapeutic purposes, or as a breeding cell line for animal resistance to H9N2 influenza virus.

[0029] Eighthly, the present invention provides the application of the TRIM25 gene / protein as a target in screening or producing H9N2 influenza virus replication enhancers, wherein the enhancer targets the TRIM25 gene / protein to promote the expression of the TRIM25 gene / protein.

[0030] In a ninth aspect, the present invention provides the use of TRIM25 protein or an expression promoter thereof in the preparation of replication enhancers for H9N2 influenza virus or production enhancers for H9N2 influenza virus vaccines.

[0031] In a tenth aspect, the present invention provides the application of TRIM25 gene overexpression cell lines as production cell lines for H9N2 influenza virus or vaccines.

[0032] The beneficial effects of this invention are: ① This invention first discovered that inhibiting or silencing the host TRIM25 gene can suppress the replication of H9N2 influenza virus, and can be used as a target for preparing drugs that inhibit the replication of H9N2 influenza virus; ② This invention provides an sgRNA targeting the TRIM25 gene, which can specifically target the TRIM25 gene, and can achieve the knockout of the TRIM25 gene in host cells by combining it with CRISPR-Cas9 technology; by delivering the mRNA sequence of the TRIM25 gene-targeting sgRNA and the Cas9 protein into animals through a drug carrier, the TRIM25 gene can be inhibited, thereby inhibiting the infection of H9N2 influenza virus; ③ This invention provides a method for delivering the sgRNA using CRISPR-Cas9 technology. The method of transfecting host cells to construct a cell line with loss of function of the TRIM25 gene-encoded protein, by knocking out the TRIM25 gene, obtained a cell line with resistance to H9N2 influenza virus, which can significantly inhibit the replication of H9N2 influenza virus. This provides research tools and materials for further study on the molecular mechanism of TRIM25 gene regulating the replication of pathogenic microorganisms in cells, and can also be used for animal breeding against H9N2 influenza virus; ④ This invention found that overexpression of TRIM25 can significantly promote the replication of H9N2 influenza virus. Therefore, TRIM25 or its expression promoter can be used as a replication or production enhancer of H9N2 influenza virus for the production of H9N2 influenza virus or vaccine; and the cell line overexpressing TRIM25 can be used as a production cell line for H9N2 influenza virus or vaccine. Attached Figure Description

[0033] Figure 1 Results of TRIM25 protein Western blotting in HEK293T cells with TRIM25 gene knockout;

[0034] Figure 2 TRIM25 gene knockout HEK293T cells TRIM25 sequencing identification results;

[0035] Figure 3 Results of viral titer detection in TRIM25 gene knockout HEK293T cells after H9N2 challenge. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of this invention clearer, the various embodiments of this invention will be described in detail below with reference to the accompanying drawings. However, those skilled in the art will understand that many technical details have been provided in the embodiments of this invention to facilitate a better understanding of this application. However, the technical solutions claimed in this application can be implemented even without these technical details and various variations and modifications based on the following embodiments.

[0037] definition

[0038] The term "gene targeting" refers to targeted transgenic technology that uses site-specific homologous recombination of DNA to modify the genetic information of cells or organisms. This mainly includes gene knockout, gene inactivation, gene knock-in, point mutation, deletion mutation, and large-segment deletion of the chromosome. "Gene knockout" specifically refers to the inactivation of a specific target gene through homologous recombination. This invention uses gene knockout technology to knock out the TRIM25 gene in host cells, obtaining a monoclonal cell line with loss of TRIM25 gene-encoded protein function that can inhibit viral replication after H9N2 infection. This invention can also successfully construct a monoclonal cell line with loss of TRIM25 gene-encoded protein function by mutating or deleting a segment of the TRIM25 gene in host cells, resulting in a frameshift mutation in the TRIM25 gene-encoded protein.

[0039] The term "sgRNA" stands for guide RNA, which refers to a small non-coding RNA that guides the insertion or deletion of uridine residues into the kinetoplastid during RNA editing.

[0040] This invention synthesizes sgRNA targeting the TRIM25 gene, and further prepares sgRNA oligonucleotides targeting the TRIM25 gene by adding CACC sticky ends to the 5' end of the forward sequence of the sgRNA fragment and AAAC sticky ends to the 5' end of the reverse sequence, and then anneals them into double-stranded fragments.

[0041] This invention, based on direct targeted splicing of the TRIM25 gene, utilizes a CRISPR-Cas9 combined with a method for specifically knocking out the TRIM25 gene. Taking human HEK293T cells as an example, the TRIM25 gene was knocked out, providing a strategy for the prevention or treatment of H9N2. Although this invention only knocked out the TRIM25 gene in HEK293T cells, obtaining a gene knockout cell with H9N2 resistance, the method described in this invention can be extrapolated and extended to TRIM25 gene knockout in other animal cells to construct gene knockout cells with TRIM25 resistance.

[0042] The CRISPR-Cas9 system achieves targeted gene recognition and cleavage through sgRNA and Cas9. sgRNA determines the targeting and cleavage activity of Cas9. This invention aims to utilize CRISPR-Cas9 gene editing technology to accurately and efficiently knock out the TRIM25 gene by screening for sgRNA sequences targeting the TRIM25 gene in vitro and in vivo. The goal is to obtain a TRIM25 gene knockout monoclonal cell line that inhibits H9N2 virus replication, thereby providing a new strategy for the prevention or treatment of H9N2 infection.

[0043] Using CRISPR-Cas9 gene editing technology, Cas9 protein is guided by sgRNA targeting the TRIM25 gene to bind to a specific sequence position in the TRIM25 gene to cut the DNA double strand, causing a double-strand break. Under the action of the cell's own repair mechanism, random mutations are generated. Mutations such as nucleotide deletion or insertion will cause changes in the gene's reading frame, ultimately achieving the goal of losing the function of the gene-encoded protein and obtaining a cell line with the loss of gene-encoded protein function.

[0044] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent companies.

[0045] The plasmids used in the following examples were purchased from the Miaoling Plasmid Platform.

[0046] Cell culture: HEK293T cells were derived from humans; they were cultured in DMEM medium containing 5% fetal bovine serum (FBS) and 1% penicillin antibiotics at 37°C in an incubator containing 5% CO2.

[0047] Virus source: (H9N2, influenza A / chicken / Hong Kong / G9 / 1997) strain is preserved at the Animal Microecological Preparation Engineering Research Center of Jilin Agricultural University.

[0048] Example 1: TRIM25 gene knockout HEK293T cell line

[0049] 1. Design of sgRNA targeting the TRIM25 gene

[0050] By querying the TRIM25 gene sequence (Gene ID: 7706) in the database, the first exon segment of TRIM25 in the overlapping region of different transcripts in different transcriptomes of the genome was located for target design.

[0051] Following the CRISPR-Cas9 design principles, sgRNA was designed using the CRISPR online design website http: / / crispr.mit.edu / and named TRIM25-sgRNA. A CACC sticky terminator was added to the 5' end of the forward sequence of the sgRNA fragment, and an AAAC sticky terminator was added to the 5' end of the reverse sequence, creating the sgRNA oligonucleotide targeting the TRIM25 gene (sgRNA1-oligo). This sgRNA-oligo was synthesized by Qingke Biotechnology Co., Ltd., and its detailed sequence is shown in Table 1.

[0052] Table 1. Oligonucleotides of sgRNA targeting the TRIM25 gene

[0053]

[0054] Note: The underlined part indicates the added restriction enzyme site, and the ununderlined part is the sgRNA sequence (shown in SEQ ID NO.1-2).

[0055] 2. Construction of the sgRNA recombinant plasmid pX459-sgRNA

[0056] Enzyme digestion of the pX459 vector plasmid: The pX459 vector was digested with BbsI restriction endonuclease in a 20 μL volume: pX459 vector, 5 μL; BbsI, 1 μL; 10× Buffer, 2 μL; ddH2O, 12 μL. The digestion was carried out at 37°C for 3 h. Nucleic acid electrophoresis was then performed, and the linearized fragment containing sticky ends of the pX459 vector was purified and recovered using an Omega DNA purification kit.

[0057] To obtain double-stranded sgRNA-oligo, the synthesized two oligo DNA molecules were annealed to form double-stranded DNA. The annealing reaction system was (20 μL): TRIM25-sgRNA-F-oligo, 0.5 μL; TRIM25-sgRNA-R-oligo, 0.5 μL; ddH2O, 18 μL; Annealing Buffer (20×), 1 μL.

[0058] After briefly centrifuging the above system, incubate it in a PCR instrument for 3 min, and then allow it to cool naturally for 20 min. Take 3.5 μL of the hybridized double-stranded DNA for T4 DNA ligase ligation reaction. The reaction system is (5 μL): pX459 digested and purified fragment, 0.5 μL; double-stranded DNA, 3.5 μL; T4 DNA Ligase, 0.5 μL; 10×T4 DNA Ligase Buffer, 0.5 μL.

[0059] The ligation product was directly transformed into highly competent DH5α *E. coli* cells. The transformation procedure was as follows: 50 μL of highly competent DH5α *E. coli* cells were mixed with 500 ng of the ligation product and placed on ice for 30 min. The cells were then heat-shocked in a 42℃ water bath for 45 s, followed by an ice bath for 2 min. 500 mL of antibiotic-free LB medium was added, and the cells were incubated at 37℃ and 220 rpm for 1 h. The revived bacterial culture was centrifuged at 4000 rpm at room temperature for 5 min, and 400 μL of supernatant was discarded. The remaining supernatant and the precipitated bacterial cells were thoroughly resuspended, and the transformed *E. coli* were spread onto ampicillin-resistant LB agar plates using a spreader. The plates were incubated at 37℃ for 12 h, and growth was observed.

[0060] Single colonies were selected and cultured in LB broth containing ampicillin resistance for 12 hours. Plasmids were extracted using the Omega plasmid extraction kit and sequenced for verification. The sequence obtained from the constructed plasmid was compared with the original vector using TRC universal primers, and the results were consistent with expectations. This indicates that the plasmid pX459-TRIM25-sgRNA expressing sgRNA was successfully constructed.

[0061] 3. Cell transfection

[0062] HEK293T cells were cultured in DMEM medium containing 5% FBS and 1% penicillin-dextrose antibodies. When the cells were stable and in good condition after 2-3 passages, they were digested and seeded into 6-well plates. Transfection was performed when cell confluence reached 80%. 2 μg of the constructed recombinant plasmid and 4 μL of Lipofectamine 2000 transfection reagent were diluted with an equal volume of DMEM and incubated for 5 min. The two solutions were then mixed in equal volumes and incubated for 5 min. All these processes were performed at room temperature. Finally, the mixture was transfected into HEK293T cells and cultured at 37°C in a 5% CO2 incubator for 24 h. Cells were then treated with puromycin medium at a concentration of 2 μg / mL for 2-3 days to screen for transfected positive cells. Cell counting was then performed, and several hundred positive cells were seeded into 96-well plates to obtain single-cell clones.

[0063] (1) TRIM25-KO Western blotting verification

[0064] HEK293T cells (wild-type cells) that have not undergone gene editing were used as a negative control. Wild-type (WT) cell lines and knockout candidate cell lines were cultured separately. After the cells reached confluence, they were harvested. The cells were lysed with a 1:1 mixture of RIPA and 2×SDS-PAGE Buffer, transferred to 1.5 mL EP tubes, incubated at 100°C for 10 min, and centrifuged at 12000 rpm for 1 min. The supernatant was then used for polyacrylamide gel electrophoresis. After the procedure, the membrane was blocked with TBST containing 5% skim milk for 1 hour; it was then incubated overnight at 4°C with primary antibodies (TRIM25 antibody and GAPDH antibody), and washed three times with 1×TBST for 5 minutes each time; secondary antibodies (HRP-conjugated goat anti-rabbit secondary antibody and HRP-conjugated goat anti-mouse secondary antibody) were incubated on a shaker at room temperature for 1 hour, and washed three times with 1×TBST for 5 minutes each time. The luminescent substrate ECL developing solution was then added to the nitrocellulose membrane, and the membrane was exposed and developed to verify the protein expression level of the TRIM25 gene knockout HEK293T cell line. The results are as follows: Figure 1 As shown, TRIM25 protein was not expressed in the TRIM25-KO HEK293T cell line.

[0065] (2) Identification of TRIM25 knockout cell lines by sequencing

[0066] mRNA was extracted from the TRIM25 gene knockout HEK293T cell line (TRIM25-KO), which does not express TRIM25 protein. The TRIM25 target sequence was amplified by RT-PCR and sent to Qingke Biotechnology Co., Ltd. for sequencing. Figure 2 As shown, the TRIM25-KO sequencing results indicate that 14 effective base deletions occurred in the amplified fragment, resulting in a frameshift mutation. Combined with the Western blotting results, no TRIM25 protein expression was detected, indicating that the TRIM25 gene knockout HEK293T cell line was successfully constructed.

[0067] Example 2: Effect of TRIM25 gene knockout on H9N2 replication in HEK293T cell line

[0068] Wild-type HEK293T cells were passaged normally and then transfected with an empty vector or an overexpression of the TRIM25 plasmid. Similarly, HEK293T cells with TRIM25 gene knockout and wild-type HEK293T cells were passaged normally and then seeded into cell culture dishes and inoculated with H9N2 influenza virus. Finally, influenza virus titration assays were used to detect H9N2 influenza virus replication.

[0069] The results are as follows Figure 3 As shown, vector represents wild-type HEK293T cells transfected with the empty vector, and TRIM25 represents HEK293T cells transfected with the TRIM25 plasmid. + / + Wild-type HEK293T cells, TRIM25 - / - The TRIM25 gene knockout HEK293T cells described in this application were used. Results showed that wild-type HEK293T cells overexpressing the TRIM25 gene (TRIM25) exhibited stronger viral replication capacity after inoculation with H9N2 influenza virus than normal wild-type HEK293T cells; and TRIM25 gene knockout HEK293T cells (TRIM25...) - / - The viral replication capacity of H9N2 influenza virus inoculated cells was weaker than that of wild-type HEK293T cells.

[0070] The above results indicate that HEK293T cells with the TRIM25 gene knocked out, obtained through gene editing technology, can significantly inhibit the replication of H9N2 influenza virus and exhibit resistance to H9N2 influenza virus. Therefore, the constructed loss-of-function cells encoding the TRIM25 gene protein can be used for breeding animals resistant to H9N2 influenza virus.

[0071] In summary, this invention successfully constructed a cell line with loss of function of the TRIM25 gene-encoded protein using CRISPR-Cas9 technology. However, this invention is not limited to CRISPR-Cas9 technology. Based on this invention, other technical means can also be used to induce loss of function of the TRIM25 gene-encoded protein to obtain cell lines with loss of TRIM25 gene function. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of this invention should be considered equivalent substitutions and are included within the scope of protection of this invention.

Claims

1. The application of a TRIM25 gene / protein expression inhibitor in the preparation of drugs for the prevention or treatment of H9N2 influenza virus infection; wherein the inhibitor is an SgRNA targeting the TRIM25 gene / protein, and the sequence of the SgRNA targeting the TRIM25 gene / protein is as follows: TRIM25-sgRNA-F: 5'-GGGAGCCACCCGCCGACGTC-3'; TRIM25-sgRNA-R: 5'-GACGTCGGCCGGGTGGCTCCC-3'.

2. A sgRNA that specifically targets the TRIM25 gene, characterized in that, The sequence of the sgRNA is as follows: TRIM25-sgRNA-F: 5'-GGGAGCCACCCGCCGACGTC-3'; TRIM25-sgRNA-R: 5'-GACGTCGGCCGGGTGGCTCCC-3'.

3. The use of the sgRNA as described in claim 2 in the preparation of drugs for the prevention or treatment of H9N2 influenza virus infection.

4. Application of TRIM25 protein in the preparation of replication enhancers for H9N2 influenza virus or production enhancers for H9N2 influenza virus vaccines.

5. Application of TRIM25 gene overexpression cell lines as production cell lines for H9N2 influenza virus or vaccines.

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