Application of monensin in preparation of anti-coronavirus drugs

Abstract

The application discloses application of monensin in preparation of anti-coronavirus drugs, relates to the technical field of new monensin purposes, and the coronavirus has a ribosome frameshift translation regulation mechanism, monensin is used for inhibiting ribosome frameshift, and then inhibiting the coronavirus proliferation. For feline infectious peritonitis virus, the effective concentration of monensin is 0.1 muM-10 muM. For canine coronavirus, the effective concentration of monensin is 0.5 muM-10 muM. The application has the following advantages: small drug resistance risk; low effective concentration; and convenient administration.

Description

Technical Field

[0001] This invention relates to the field of novel uses of monensin, and more particularly to the application of monensin in the preparation of anti-coronavirus drugs. Background Technology

[0002] Feline infectious peritonitis (FIP) is a fatal disease caused by mutant strains of feline coronavirus (FCoV). Its pathogenesis involves the synergistic effect of viral subtype transformation and immunopathological responses. FCoV is classified into type I and type II based on antigenicity, and each type is further divided into enteric type (FECV) and lethal type (FIPV) based on pathogenicity. Approximately 90% of FCoV infections present as the FECV subtype, causing only self-limiting gastrointestinal or respiratory symptoms. About 10% of infected individuals undergo viral mutation, transforming into the FIPV subtype. This mutant strain spreads systemically through infection of the mononuclear macrophage system, ultimately causing granulomatous inflammation around blood vessels in the abdominal organs, kidneys, and central nervous system. Its core mechanism is a type III hypersensitivity reaction triggered by viral antigen-antibody complex deposition, leading to increased vascular permeability and multiple organ dysfunction syndrome. Type I strains include FECV-UCD, FIPV-I-UCD3, FIPV-I-UCD4, and FIPV-I-TN406. These strains are difficult to culture, proliferate only in macrophages, and exhibit weak serological cross-reactivity with canine coronavirus (CCoV). Type II strains include FIPV-II-NOR15, FEPV-II-79–183, and FIPV-II-79–1146. These are produced by recombination of Type I and CCoV, readily replicate in various cell lines, and have high viral yields. Type I is more prevalent in natural infections, but research has largely focused on Type II. The two strains have different receptors: Type II depends on feline aminopeptidase-N (fAPN), while Type I depends on feline dendritic cell-specific adhesion molecule (fDC-SIGN). The complexity of FIPV strains makes the development of antiviral drugs encompassing all FIPV strains extremely challenging. Feline infectious peritonitis (FIP) was once considered an incurable disease, and currently, there are no officially approved specific drugs against FIP ​​virus in China. Although the emergence of antiviral drugs such as GS-441524 and Molnupiravir in recent years has brought some hope for the treatment of FIP, the long-term efficacy and drug resistance risks of these drugs still require further research. Therefore, it is necessary to develop novel specific drugs against FIP ​​virus to address the current predicament.

[0003] Canine coronavirus (CCoV) is a highly contagious, globally prevalent pathogenic virus in dogs, causing intestinal disease and posing a threat to the canine industry and canine health. Both CCoV and FCoV belong to the alpha coronavirus family. Although both are alpha coronaviruses, their gene sequences and host specificities differ significantly: CCoV primarily infects canine intestinal epithelial cells, while FCoV tends to target the mononuclear phagocyte system in cats, and their pathogenic mechanisms also differ. CCoV is mainly transmitted through ingestion of infected feces or contact with contaminated objects; crowded conditions and poor hygiene accelerate transmission. The incubation period is 1 to 4 days, and the course of the disease is typically 2 to 10 days. Secondary bacterial or parasitic infections may occur. Recovered dogs can carry the virus for up to 6 months. The CCoV infection rate in Chinese dogs is as high as 33%. Symptoms of CCoV infection are often asymptomatic, but in severe cases, they manifest as sudden diarrhea, lethargy, and loss of appetite. The feces are orange and may contain blood or mucus, easily confused with parvovirus. CCoV infection is significantly associated with canine diarrhea and has become a common health concern for veterinarians and dog owners in China. Currently, there are no specific antiviral drugs for CCoV, and treatment is mainly symptomatic and supportive, with common regimens including antibiotics, antiemetics, and antidiarrheals. There is an urgent need to develop a specific antiviral drug for CCoV.

[0004] Programmed -1 frameshifting (-1 PRF) is a translational regulatory mechanism commonly used in coronaviruses but rare in mammals, which regulates the expression ratio of two proteins on mRNA. This process occurs when the ribosome encounters a specific mRNA signal consisting of a frameshift site and a downstream stimulating structure. Its efficiency is influenced by both ribosome conformational changes and the dynamic mechanical properties of the stimulating structure. Most -1 PRFs occur on a seven-nucleotide slip sequence with an X-XXY-YYZ arrangement (where XXX and YYY represent three identical nucleotides separated by hyphens). This arrangement allows for the selection of different codons within the slip sequence and minimizes the differences between codons after transitioning to the -1 reading frame (XXX-YYY-Z).

[0005]

[0006] Monensin, also known as rumensin, is produced by *Streptomyces cinnamonensis*. Monensin is a mixture containing multiple homologues, including A, B, C, and D. Monensin A and monensin B are the main components. Monensin A is an orally active ionocarrier antibiotic that mediates Na+ ionocarrier transport. + / H +Exchange plays a role. Monensin is currently widely used in veterinary medicine and has been developed as an anticoccidial drug and growth promoter for ruminants. However, the role of monensin in combating feline infectious peritonitis virus and canine coronavirus infection remains under investigation. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides the application of monensin in the preparation of anti-coronavirus drugs, and explores the effect of monensin on inhibiting feline infectious peritonitis virus and canine coronavirus.

[0008] In order to achieve the objective of this invention, the following solution is proposed:

[0009] An anti-coronavirus drug, wherein the sole active ingredient of the drug is monensin, and the coronavirus is feline infectious peritonitis virus or canine coronavirus.

[0010] Furthermore, when the coronavirus is feline infectious peritonitis virus, the effective concentration of monensin is 0.1 μM-2 μM.

[0011] Furthermore, when the coronavirus is a canine coronavirus, the effective concentration of monensin is 0.5 μM-2 μM.

[0012] The application of monensin in the preparation of anti-coronavirus drugs, wherein monensin is used as the sole active ingredient of the drug to inhibit the proliferation of feline infectious peritonitis virus or canine coronavirus.

[0013] The beneficial effects of this invention are as follows:

[0014] 1. Monensin has an inhibitory effect on the ribosomal frameshift of feline infectious peritonitis virus and canine coronavirus, thereby inhibiting the proliferation of feline infectious peritonitis virus and canine coronavirus.

[0015] 2. Monensin has a low risk of drug resistance. Ribosome frameshift RNA elements are functional RNA elements and are highly conserved in evolution. Monensin targets ribosomal frameshift elements, which makes its drug resistance risk lower than that of current drugs that target proteins.

[0016] 3. Monensin has a low effective concentration. Ribosome frameshifting during genomic RNA translation is a vulnerable link in the viral life cycle of FIPV and CCoV. Viruses are very sensitive to changes in ribosome frameshifting efficiency. Therefore, monensin, which targets ribosome frameshifting elements, has a very low effective concentration.

[0017] 4. Monensin is easy to administer. Monensin is a food additive that can be ingested orally by being added to food. Attached Figure Description

[0018] Figure 1This is a luciferase screening system, in which, Figure 1 (a) is a diagram of the construction strategy for dual-luciferase reporter gene vectors; Figure 1 (b) is a diagram of compound screening strategies; Figure 1 (c) is a diagram showing the effect of monensin A on dual-luciferase reporter cells;

[0019] Figure 2 For fluorescent protein screening systems, among which, Figure 2 (a) is a diagram illustrating the strategy for constructing dual-fluorescent protein reporter gene vectors; Figure 2 (b) is a diagram of compound screening strategies; Figure 2 (c) is a diagram showing the effect of monensin A on cells with dual fluorescent protein reporter genes;

[0020] Figure 3 This is a cytopathic image showing the inhibition of FIPV proliferation by monensin A.

[0021] Figure 4 A cytopathic effect diagram showing the inhibition of CCoV proliferation by monensin A;

[0022] Figure 5 The results are plotted for EC50, CC50, and TCID50; among them, Figure 5 (a) is a graph showing the CC50 and EC50 results of monensin against FIPV virus; Figure 5 (b) is a graph showing the CC50 and EC50 results of monensin against CCoV virus; Figure 5 (c) is a graph showing the TCID50 calculation results of monensin inhibiting the proliferation of FIPV and CCoV viruses. Detailed Implementation

[0023] The experimental method is as follows:

[0024] We used in vitro cell culture to screen FDA-approved drug libraries, 5K cytoskeleton diversity libraries, natural product libraries, and traditional Chinese medicine monomer libraries to investigate whether each drug inhibits the ribosomal frameshift process of FIPV and cCoV. First, we constructed a dual-luciferase reporter gene lentiviral vector containing the FIPV and cCoV viral genome sliding sequences, and then tested the effects of different drugs in the feline kidney cell line F81 and canine kidney cell line MDCK. The initial screening concentration was 10 μM.

[0025] Lentiviral backbone vectors, lentiviral packaging vectors pspax2 and PMD2.G were preserved in the laboratory. Ribosomal frameshift elements of FIPV and CCoV were synthesized by Qingke. Feline kidney cell line F81 and canine kidney cell line MDCK were preserved in the laboratory.

[0026] Construction of luciferase reporter gene vector:

[0027] (1) Synthesis of FIPV and CCoV viral ribosomal frameshift region gene sequences. The lentiviral backbone vector was double-digested with EcoRI and BamHI. Digestion conditions: 37℃ for 15 minutes. After digestion, the digestion products were recovered by nucleic acid electrophoresis. For the synthesized FIPV viral ribosomal frameshift region gene sequence, denaturation was first performed at 95℃ for 10 minutes and annealing at 72℃ for 30 seconds. Then, the annealing product was mixed with the digestion product at a ratio of annealing product: digestion product = 3:1. 10 μL of T4 ligase was added to the mixture, and ligation was carried out in a 16℃ constant temperature metal bath for 16 hours. Plasmid transformation of competent cells: Competent cells were removed from a -80°C freezer and thawed on ice. A lentiviral vector containing the ribosomal frameshift region gene sequence of FIPV or CCoV virus was added to every 100 μL of competent cells and mixed thoroughly. The mixture was then placed on ice for 30 minutes. The mixture was then placed in 42°C water for 90 seconds for heat shock. After heat shock, the mixture was cooled on ice for 10 seconds. The cooled mixture was transferred to a solid LB agar plate, and the liquid was evenly spread on the surface of the solid LB medium using a glass rod. The plate was then inverted and incubated at 37°C for 16 hours. After incubation, single-clone plaques were picked up with a pipette tip and inoculated into liquid LB medium. After incubation at 37°C for 8 hours, the plates were sent to the company for sequencing. Single clones with correct sequencing results were expanded and plasmids were extracted.

[0028] (2) Through homologous recombination, such as Figure 1 As shown in (a), Renal luciferase was constructed upstream of the ribosomal frameshift region, and firefly luciferase was constructed downstream of the ribosomal frameshift region. The specific method is as follows: The gene sequences of Renal luciferase and firefly luciferase were amplified by PCR, and homologous arms were added upstream and downstream of the sequences, respectively. The vector obtained in step (1) was subjected to PCR to obtain a linearized vector. The gel recovery products of Renal luciferase and firefly luciferase, as well as the gel recovery product of the linearized vector, were obtained by nucleic acid electrophoresis and gel recovery. Homologous recombination: The gel recovery products of Renal luciferase and firefly luciferase were mixed with the gel recovery product of the linearized vector at a mass ratio of 3:1, and 2 μL of homologous recombination enzyme was added. The mixture was reacted at 37°C for 15 minutes.

[0029] (3) Transform competent cells with plasmids, select single clones and sequence them, and amplify and extract plasmids from viral backbone vectors with correct sequencing. The transformation of competent cells and extraction steps are the same as in step (1).

[0030] Establishing a luciferase reporter gene screening cell system:

[0031] (1) The viral backbone vector containing dual luciferase and ribosomal frameshift region, pspax2, and PMD2.G packaging vector were mixed with PEI in a volume ratio of 4:2:1. 70 micrograms of PEI were added for every 35 micrograms of DNA and transfected into 293T cells.

[0032] (2) Collect the culture supernatant of 293T cells 48h and 72h after transfection with viral plasmid, respectively. After centrifuging at 12000g for 10min to remove cell impurities, collect lentivirus particles by cesium chloride gradient centrifugation.

[0033] (3) Lentiviral particles were added to F81 cells and MDCK cells. After 7 days of infection, positive cells were screened by Puro. Cells were digested with trypsin to prepare single-cell suspensions, and single-clone sorting was performed by flow cytometry.

[0034] (4) Genotyping of cultured monoclonal cells and expansion culture of positive clones to obtain F81 cells and MDCK cells (CMV-Renilla-framshift-Firefly) containing ribosomal frameshift regions.

[0035] Drug screening based on luciferase reporter genes, such as Figure 1 As shown in (b):

[0036] (1) F81 cells and MDCK cells containing ribosomal frameshift regions were mixed at a ratio of 1:1 and cultured together in a 96-well plate. Different test compounds were added to each well, with a drug concentration of 10 μM.

[0037] (2) After culturing for 8 hours, the cells were lysed, firefly luciferase substrate was added to the lysate, and the luminescence value was detected by an enzyme-linked immunosorbent assay (ELISA) reader.

[0038] (3) Add the Renaissance luciferase substrate and detect the luminescence value using an enzyme-linked immunosorbent assay (ELISA) reader.

[0039] (4) The ratio of firefly luciferase to Renilla luciferase is used as a reference for ribosome frameshift efficiency. A higher ratio indicates higher ribosome frameshift efficiency, while a lower ratio indicates lower efficiency. In other words, a lower ratio indicates a higher efficiency of the drug in inhibiting ribosome frameshift, and thus a better inhibitory effect on the process. Figure 1 As shown in (c), after the addition of monensin A, the relative firefly luciferase activity of the binary mixed screening cells decreased from about 60% to less than 10%.

[0040] Construction of fluorescent protein reporter gene vector:

[0041] (1) Synthesis of FIPV and CCoV viral ribosomal frameshift region gene sequences. The lentiviral backbone vector was double-digested with EcoRI and BamHI. Digestion conditions: 37℃ for 15 minutes. After digestion, the digestion products were recovered by nucleic acid electrophoresis. For the synthesized FIPV viral ribosomal frameshift region gene sequence, denaturation was first performed at 95℃ for 10 minutes and annealing at 72℃ for 30 seconds. Then, the annealing product was mixed with the digestion product at a ratio of annealing product: digestion product = 3:1. 10 μL of T4 ligase was added to the mixture, and ligation was carried out in a 16℃ constant temperature metal bath for 16 hours. Plasmid transformation of competent cells: Competent cells were removed from a -80°C freezer and thawed on ice. A lentiviral vector containing the FIPV viral ribosomal frameshift region gene sequence was added at a ratio of 10 μL of competent cells to 10 μL of plasmid (the plasmid is a lentiviral vector containing the FIPV viral ribosome frameshift region gene sequence). The mixture was incubated on ice for 30 minutes, then the cell-plasmid mixture was placed in 42°C water for 90 seconds for heat shock. After heat shock, the cell-plasmid mixture was cooled on ice for 10 seconds. The cooled mixture was transferred to a solid LB agar plate, and the liquid was evenly spread on the surface of the solid LB medium using a glass rod. The plate was then inverted and incubated at 37°C for 16 hours. After incubation, single-clone plaques were picked up with a pipette tip and inoculated into liquid LB medium. After incubation at 37°C in a shaker for 8 hours, the plates were sent for sequencing. Single clones with correct sequencing results were expanded and plasmids were extracted.

[0042] (2) Through homologous recombination, such as Figure 2 As shown in (a), ubiquitin-green fluorescent protein (Ub-GFP) was constructed upstream of the ribosomal frameshift region, and red fluorescent protein (RFP) was constructed downstream of the ribosomal frameshift region. The specific method is as follows: the Ub-GFP and RFP gene sequences were amplified by PCR, and homologous arms were added upstream and downstream of the sequences, respectively. The vector obtained in step (1) was subjected to PCR to obtain a linearized vector. The gel recovery products of Ub-GFP and RFP and the linearized vector were obtained by nucleic acid electrophoresis and gel recovery. Homologous recombination: the gel recovery products of Ub-GFP and RFP and the gel recovery product of linearized vector were mixed at a mass ratio of 3:1, and 2 μL of homologous recombinase was added. The mixture was reacted at 37°C for 15 minutes.

[0043] (3) Transform competent cells with plasmids, select single clones and sequence them, and amplify and extract plasmids from viral backbone vectors with correct sequencing. The transformation and extraction steps are the same as steps (1) in the fluorescent protein reporter gene vector construction process.

[0044] Establishing a fluorescent protein reporter gene screening cell system:

[0045] (1) The viral backbone vector containing dual fluorescent protein and ribosomal frameshift region, pspax2, and PMD2.G packaging vector were mixed with PEI in a volume ratio of 4:2:1. 70 micrograms of PEI were added for every 35 micrograms of DNA and transfected into 293T cells.

[0046] (2) Collect the culture supernatant of 293T cells 48h and 72h after transfection with viral plasmid, respectively. After centrifuging at 12000g for 10min to remove cell impurities, collect lentivirus particles by cesium chloride gradient centrifugation.

[0047] (3) Lentiviral particles were added to F81 cells and MDCK cells. After 7 days of infection, positive cells were screened by Puro. Cells were digested with trypsin to prepare single-cell suspensions, and single-clone sorting was performed by flow cytometry.

[0048] (4) Genotyping of cultured monoclonal cells and expansion culture of positive clones to obtain F81 cells and MDCK cells (CMV-Ub-GFP-framshift-RFP) containing FIPV-1 and CCoV virus Frameshift region.

[0049] Drug screening based on fluorescent protein reporter genes, such as Figure 2 As shown in (b):

[0050] (1) F81 cells containing ribosomal frameshift regions and MDCK fluorescent protein reporter gene cells were mixed in a 1:1 ratio and cultured together in a 96-well plate. After the cells were plated for 24 hours, different test compounds were added to the plates. The drug concentration was 10 μM, and autofluorescent compounds were excluded.

[0051] (2) After culturing for 8 hours, MG132 was added, and after culturing for 4 hours, the changes in fluorescence signal were observed under a high-content fluorescence microscope.

[0052] (3) The ratio of RFP to GFP is used as a reference for ribosome frameshift efficiency. A higher ratio indicates higher ribosome frameshift efficiency, while a lower ratio indicates lower efficiency. In other words, a lower ratio indicates a higher efficiency of the drug in inhibiting the ribosome frameshift process, and thus a better inhibitory effect. Figure 2 As shown in (c), similar to the results of dual-luciferase reporter gene detection, after the addition of monensin A, the relative fluorescence intensity of the relative red fluorescent protein in the binary mixed screening cells decreased from about 60% to less than 10%.

[0053] In vitro antiviral activity:

[0054] CRFK (feline kidney) cells, A-72 canine tumor fibroblasts, FIPV virus (FIPV 79-1146), and canine coronavirus (CCoV 1-71) were preserved in our laboratory. All experiments were performed in a P2 laboratory, and all procedures complied with biosafety management protocols. CRFK and A-72 cells were cultured separately in DMEM high-glucose medium. After cell confluence reached 90%, cells were trypsinized and seeded in six-well plates at 200,000 cells / well, incubated overnight at 37°C. FIPV and CCoV viruses (MOI=0.01) were added separately. After 2 hours of virus adsorption, the medium was replaced with normal medium. A compound was added to a final concentration of 0.2 μM. Pure cell negative control wells and virus positive control wells were set up. Cell pathogenesis in each well was observed after 48 hours. Changes in viral load were detected using quantitative real-time PCR.

[0055] Table 1 Primer Record for Quantitative Real-Time PCR Detection

[0056]

[0057] Figure 3 The results show the cytopathic effect of monensin on FIPV proliferation. Without the virus, CRFK cells exhibited a spindle-shaped morphology and good growth. After FIPV infection, the solvent-treated group (DMSO group) showed significant cytopathic effects. With the addition of 1 μM monensin, the cell state was close to that of the virus-free group. Although sporadic cytopathic effects were also observed in the 0.1 μM monensin-treated group, the number of cytopathic effects was significantly reduced, and the overall cell state was better than the solvent control group.

[0058] Figure 4 The results show the cytopathic effect of monensin on the inhibition of CCoV proliferation. The results indicate that A-72 cells grew well without the addition of virus. After CCoV infection, the solvent-treated group (DMSO group) showed significant cytopathic effects. Although the 0.1 μM monensin treatment group did not show significant cytopathic effect remission, the 1 μM monensin treatment group showed less cytopathic effect, and the cell state was similar to the virus-free group.

[0059] CC50 calculation: Monensin at concentrations of 0.01 μM, 0.1 μM, 0.2 μM, 0.5 μM, 1 μM, 2 μM, 5 μM, 10 μM, 20 μM, 50 μM, and 100 μM (with 5 replicates) was added to the cell culture supernatant. The cells were cultured at 37°C and 5% CO2 for 48 h. Cell viability was detected using a CCK8 assay kit, and the CC50 values ​​of monensin in CRFK and A-72 cells were calculated using GraphPad software.

[0060] EC50 calculation: Monensin at concentrations of 0.01 μM, 0.1 μM, 0.2 μM, 0.5 μM, 1 μM, 2 μM, 5 μM, 10 μM, 20 μM, 50 μM, and 100 μM (with 5 replicates) was added to the culture supernatant of CRFK and A-72 cells, along with FIPV and CCoV viruses (MOI=0.01). The cells were cultured at 37°C and 5% CO2 for 48 h. Bright-field images were taken under a microscope to obtain cytopathic effect data. The EC50 values ​​of monensin inhibiting FIPV and CCoV in CRFK and A-72 cells were calculated using GraphPad software.

[0061] TCID50 calculation: Virus was collected from cell supernatant after 48 h of treatment with 0.5 μM monensin. The virus was serially diluted using cell culture medium, each time by a 10-fold gradient. -1 Dilute to 10 -8 The virus was seeded into 96-well plates, one vertical row (8 wells) for each dilution. Cells were seeded into the 96-well plates containing the virus solution at a density of 1 × 10⁶ cells / well. 4 / well. After 48 hours of incubation, bright-field images were taken using a microscope. The number of wells with CPE at each dilution was counted, and the cumulative CPE percentage was calculated. TCID50 was calculated using the Karber method.

[0062] Figure 5 (a) Shows the CC50 and EC50 results of monensin against FIPV virus. The curves showing the changes in monensin toxicity and anti-FIPV efficacy in CRFK cells (n=5) are shown. The blue curve represents the increasing inhibition rate of monensin against FIPV, indicating that 0.1 μM monensin exhibits the ability to inhibit FIPV proliferation, and 0.5 μM monensin shows an inhibition rate close to 100%. The red curve shows the changes in monensin cytotoxicity in CRFK cells, indicating that cytotoxicity only occurs at concentrations greater than 10 μM. The calculated CC50 value of monensin in CRFK cells is 31.02 μM, and its anti-FIPV EC50 value is 0.067 μM, indicating a relatively large safety window.

[0063] Figure 5(b) Shows the CC50 and EC50 results of monensin against CCoV virus. The curves showing the changes in monensin toxicity and anti-CCoV efficacy in A-72 cells (n=5) are shown. The blue curve represents the increasing inhibition rate of monensin against CCoV, indicating that 0.5 μM monensin showed an inhibition rate close to 100%. The red curve shows the change in monensin cytotoxicity in A-72 cells, indicating that monensin only exhibits cytotoxicity at concentrations greater than 10 μM. The calculated CC50 value of monensin in A-72 cells was 28.78 μM, and its anti-CCoV EC50 value was 0.177 μM, indicating a relatively large safety window.

[0064] Figure 5 (c) TCID50 calculation results for monensin's inhibition of FIPV and CCoV viral replication. The left side shows the FIPV results, where 0.5 μM monensin reduced FIPV viral load by 4 titers (Log10). The right side shows the CCoV results, indicating that 0.5 μM monensin reduced CCoV viral load by 2 titers (Log10).

[0065] The above embodiments are only used to illustrate the technical concept and features of the present invention, and are not intended to be unique or to limit the present invention. Those skilled in the art should understand that various changes or equivalent substitutions made to the present invention without departing from its scope are all within the protection scope of the present invention.

Claims

1. Use of monensin in the preparation of a drug against coronavirus, characterized in that, The monensin is the only active ingredient of the medicament, and the coronavirus is feline infectious peritonitis virus or canine coronavirus. The monensin is the only active ingredient of the medicament, and the coronavirus is feline infectious peritonitis virus or canine coronavirus.

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