Application of nucleoside analogue in preparation of drugs for treating wetland virus diseases

Abstract

The invention discloses an application of a non-ester nucleoside analogue or a pharmaceutically acceptable salt thereof or a composition containing the non-ester nucleoside analogue in preparation of a medicine for treating wetland virus diseases, and particularly, the nucleoside analogue is preferably ribavirin and favipiravir, has an obvious inhibition effect on WELV (Wetland Virus Virus) and is dose-dependent; the method has an excellent application prospect in treatment of wetland virus diseases.

Description

Technical Field

[0001] The invention discloses pharmaceutical use of a nucleoside analogue and belongs to the technical field of drug preparation. Background Art

[0002] Ticks are important vectors that often transmit pathogens by biting and sucking blood. Ticks can carry more than 200 pathogens, including viruses, bacteria, spirochetes, protozoa, etc., including many important pathogens of natural epidemic diseases and zoonoses. The viruses carried by ticks are called tick-born viruses (TBV). Currently, TBVs that can cause human and animal diseases worldwide are mainly concentrated in the Nairoviridae, Phenuiviridae, Flaviviridae, Orthomyxoviridae and Reoviridae families. Common viruses that can cause zoonotic diseases include severe fever with thrombocytopenia syndrome virus (SFTSV) and tick-borne encephalitis virus (TBEV). Wetland virus (WELV) belongs to Nairoviridae and is isolated from humans and ticks. The clinical symptoms of this virus infection are mainly characterized by non-specific clinical symptoms such as persistent fever, dizziness, headache and multiple organ dysfunction. Field investigations have found that the virus has a wide host range and the virus has been detected in horses, pigs and sheep, which has public health significance.

[0003] Ribavirin is a nucleoside analogue that was first synthesized and developed in 1972. It was initially used for respiratory syncytial virus (RSV) and hepatitis C virus (HCV) infections. Due to its broad spectrum and diverse antiviral mechanisms, it has been verified to have certain antiviral effects on Hantavirus, SFTSV and CCHFV of the Bunyaviridae family. Favipiravir is also a nucleoside analogue with broad-spectrum antiviral effects. It was originally designed as an anti-influenza virus drug. It has been verified to have certain antiviral effects on SFTSV, CCHFV and Rift Valley Fevervirus (RVFV) of the Bunyaviridae family. The purpose of the present invention is to provide a new use of a nucleoside analogue for the preparation of wetland virus therapeutic drugs. Summary of the invention

[0004] Based on the above purpose, the present invention first provides a non-ester nucleoside analog or a pharmaceutically acceptable salt thereof or a composition containing the same for use in the preparation of a drug for treating wetland viral diseases. The non-ester nucleoside analog described in the present invention refers to a nucleoside analog that does not contain an ester group, and the ester group is the functional group of the ester in the carboxylic acid derivative, and the structural formula is -COOR (R is generally an alkyl group or other non-H group). For example, in the present invention, the nucleoside analog of formula (I) and the nucleoside analog of formula (II) are compounds that do not contain an ester group.

[0005] In a preferred embodiment, the absence of an ester group refers to the absence of a propionate group or a phosphate group. In the present invention, the nucleoside analogs of the nucleoside analogs of formula (III) and formula (V) contain a propionate group, and the nucleoside analogs of the nucleoside analogs of formula (IV) contain a phosphate group.

[0006] In a more preferred embodiment, the non-ester nucleoside analog contains an amide group. The amide group is an organic compound formed by replacing the hydroxyl group in the carboxylic acid with an amino group or an amine group, and the structural formula of the amide group is -CO-NH2. In the present invention, the nucleoside analog having formula (I) and the nucleoside analog having formula (II) both contain an amide group.

[0007] In a particularly preferred embodiment, the amide-containing nucleoside analog is a nucleoside analog having formula (I): (I).

[0009] The compound with this structural formula is called Ribavirin and its molecular formula is C8H 12 N4O5, chemical name is 1-β-D-ribofuranosyl-1H-1,2,4-triazole-3-carboxamide.

[0010] Still more preferably, the nucleoside analogue of formula (I) is prepared at a dosage of 25-125 mg / kg body weight.

[0011] Preferably, the nucleoside analogue of formula (I) is prepared at a dosage of 100 mg / kg body weight.

[0012] In another particularly preferred embodiment, the amide-containing nucleoside analog is a nucleoside analog having formula (II): (II).

[0013] The compound with this structural formula is traded as Favipiravir, has a molecular formula of C5H4FN3O2, and a chemical name of 6-fluoro-3-hydroxypyrazine-2-carboxamide.

[0014] More preferably, the nucleoside analogue of formula (II) is prepared at a dosage of 75-375 mg / kg body weight.

[0015] Preferably, the nucleoside analogue of formula (II) is prepared at a dosage of 300 mg / kg body weight.

[0016] In another preferred embodiment, the drug for treating wetland viral diseases also includes a pharmaceutically acceptable carrier, and the drug is prepared in dosage forms including tablets, capsules, solutions, emulsions, suspensions, injections, lyophilized agents, sprays, and aerosols.

[0017] The non-ester nucleoside analogs ribavirin and favipiravir provided by the present invention having an amide group show a significant inhibitory effect on WELV in a dose-dependent manner, and the IC values ​​of ribavirin and favipiravir are 50They were 15.41μM and 9.6μM, respectively. Moreover, ribavirin and favipiravir themselves had no effect on cell proliferation, and had the advantages of high efficiency and low toxicity. However, sofosbuvir, remdesivir, and monaprevir, which contain ester groups, had no inhibitory effect on WELV at 24 hours, 48 ​​hours, and 72 hours of medication. The survival rate of mice treated with higher doses of ribavirin (100mpk) and favipiravir (300mpk) on the first and second days after infection was 100%, and all mice in the control group died. Lower doses of ribavirin (25mpk) and favipiravir (75mpk) can still provide most of the protection, with mouse survival rates of 85.71% and 71.42%, respectively. Administration of ribavirin 25mpk or favipiravir 75mpk can significantly reduce the viral load in the serum, liver, spleen, and lungs of mice. The disclosure of the present invention shows that ribavirin and favipiravir have excellent application prospects in the preparation of drugs for treating wetland viral diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The results of the in vitro inhibitory activity test of ribavirin at different concentrations on WELV in Vero cells were determined by real-time quantitative polymerase chain reaction (RT-qPCR) in Example 1; Figure 2 The results of the in vitro inhibitory activity test of different concentrations of Favipiravir on WELV in Vero cells were determined by real-time quantitative polymerase chain reaction (RT-qPCR) in Example 1; Figure 3 The effect of ribavirin on the proliferation of Vero cells was determined by using the CCK-8 cell proliferation-toxicity detection kit in Example 1; Figure 4 The effect of Favipiravir on Vero cell proliferation was determined by CCK-8 cell proliferation-toxicity detection kit in Example 1; Figure 5 The results of the in vitro inhibitory activity test of different concentrations of sofosbuvir on WELV at 24 hours in Vero cells were determined by real-time quantitative polymerase chain reaction (RT-qPCR) in Example 1; Figure 6 The results of the in vitro inhibitory activity test of different concentrations of sofosbuvir on WELV at 48 hours in Vero cells were determined by real-time quantitative polymerase chain reaction (RT-qPCR) in Example 1; Figure 7 The results of the in vitro inhibitory activity test of different concentrations of sofosbuvir on WELV at 72 hours in Vero cells were determined by real-time quantitative polymerase chain reaction (RT-qPCR) in Example 1; Figure 8The results of the in vitro inhibitory activity test on WELV at different concentrations of remdesivir at 24 hours in Vero cells were determined by real-time quantitative polymerase chain reaction (RT-qPCR) in Example 1; Fig. 9 The results of the in vitro inhibitory activity test on WELV at different concentrations of remdesivir at 48 hours in Vero cells were determined by real-time quantitative polymerase chain reaction (RT-qPCR) in Example 1; Fig.10 The results of the in vitro inhibitory activity test on WELV at different concentrations of remdesivir at 72 hours in Vero cells were determined by real-time quantitative polymerase chain reaction (RT-qPCR) in Example 1; Fig.11 The results of the in vitro inhibitory activity test of WELV at different concentrations of monaprevir in Vero cells at 24 hours were determined by real-time quantitative polymerase chain reaction (RT-qPCR) in Example 1; Fig.12 The results of the in vitro inhibitory activity test of WELV at different concentrations of monaprevir in Vero cells at 48 hours were determined by real-time quantitative polymerase chain reaction (RT-qPCR) in Example 1; Fig.13 The results of the in vitro inhibitory activity test of WELV at different concentrations of monaprevir in Vero cells at 72 hours were determined by real-time quantitative polymerase chain reaction (RT-qPCR) in Example 1; Fig.14 This is the survival curve of WELV-infected mice under the condition of therapeutic administration in Example 2, using ribavirin 100 mpk on the first day after infection; Fig.15 This is the survival curve of WELV-infected mice under the condition of therapeutic administration in Example 2, using ribavirin 100 mpk on the second day after infection; Fig.16 This is the survival curve of WELV-infected mice under the condition of therapeutic administration in Example 2, using 300 mpk of Favipiravir on the first day after infection; Fig.17 This is the survival curve of WELV-infected mice under the condition of therapeutic administration in Example 2, when treated with Favipiravir 300 mpk on the second day after infection; Fig.18 This is the survival curve of WELV-infected mice under the condition of therapeutic administration in Example 2, when ribavirin 25 mpk was used for treatment on the first day after infection; Fig.19This is the survival curve of WELV-infected mice under the condition of therapeutic administration in Example 2, when ribavirin 25 mpk was used for treatment on the second day after infection; Fig. 20 This is the survival curve of WELV-infected mice treated with Favipiravir 75 mpk on day 1 after infection under the condition of therapeutic administration in Example 2; Fig.21 This is the survival curve of WELV-infected mice treated with Favipiravir 75 mpk on the second day after infection under the condition of therapeutic administration in Example 2; Fig. 22 The results of serum viral load detection of WELV-infected mice under the condition of ribavirin 25 mpk treatment under the condition of therapeutic administration in Example 2; Fig.23 The results of the liver viral load test of WELV-infected mice under the condition of ribavirin 25 mpk treatment under the condition of therapeutic administration in Example 2; Fig.24 The results of the detection of spleen viral load in mice infected with WELV under the condition of ribavirin 25 mpk treatment under the condition of therapeutic administration in Example 2; Fig.25 The results of the lung viral load test of mice infected with WELV under the condition of ribavirin 25 mpk treatment under the condition of therapeutic administration in Example 2; Fig.26 The results of serum viral load detection of WELV-infected mice under the treatment condition of 75 mpk of Favipiravir under the condition of therapeutic administration in Example 2; Fig. 27 The results of liver viral load detection of WELV-infected mice under the treatment condition of 75 mpk of Favipiravir under the condition of therapeutic administration in Example 2; Fig.28 The results of the spleen viral load test of WELV-infected mice under the treatment condition of 75 mpk of Favipiravir under the therapeutic administration in Example 2; Fig.29 This is the result of detecting the lung viral load of WELV-infected mice under the treatment condition of 75 mpk of Favipiravir under the therapeutic administration in Example 2. DETAILED DESCRIPTION

[0019] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of protection defined by the claims of the present invention.

[0020] Materials and reagents Ribavirin was purchased from MedChemExpress with a purity greater than 99.96%; favipiravir was purchased from MedChemExpress with a purity greater than 99.93%; sofosbuvir was purchased from MedChemExpress with a purity greater than 99.93%; remdesivir was purchased from MedChemExpress with a purity greater than 99.95%; monaprevir was purchased from MedChemExpress with a purity greater than 99.93%.

[0021] The molecular formula of Ribavirin is C8H 12 N4O5, chemical name is 1-β-D-furanosyl-1H-1,2,4-triazole-3-carboxamide, structural formula is (I), (I)

[0022] Favipiravir, with a molecular formula of C5H4FN3O2, a chemical name of 6-fluoro-3-hydroxypyrazine-2-carboxamide, and a structural formula of (II), (II).

[0023] Sofosbuvir, molecular formula is C 22 H 29 FN3O9P, chemical name is (S)-isopropyl-2-((S)-((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)-(phenoxy)phosphoamino)propionate, structural formula is (III), (III).

[0024] The molecular formula of Remdesivir is C 27 H 35 N6O8P, chemical name is 2-methoxy-3,3-dimethyl-2-oxocycloprop-1-enyl adenine-5'-triphosphate, structural formula is (IV), (IV).

[0025] Molnupiravir, molecular formula C 13 H 19 N3O7, chemical name is {(2R,3S,4R,5R)-3,4-dihydroxy-5-[(4Z)-4-(oxime)-2-oxo-3,4-dihydropyrimidin-1(2H)-yl]oxolan-2-yl}methyl-2-methylpropanoate, structural formula is (V), (V).

[0026] The in vitro and in vivo activity tests against wetland viruses were carried out in a biosafety level 2 laboratory. The WELV strain PS1 in the following example (Zhang XA, Ma YD, Zhang YF, et al. A new orthonairovirus associated with human febrile illness. N Engl J Med. 2024 Sep 5;391(9):821-831. doi: 10.1056 / NEJMoa2313722.) can be obtained from the applicant by the public in accordance with the relevant provisions of national biosafety. The biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0027] Example 1. Inhibition of WELV replication in Vero cells by ribavirin and favipiravir 1.1 Determination of the inhibitory effect of ribavirin and favipiravir on WELV replication in vitro by real-time quantitative polymerase chain reaction (RT-qPCR) 1) Vero cell culture: African monkey kidney cells (Vero) were evenly plated in a 24-well plate, with 100,000 cells per well, cultured in DMEM + 2% FBS medium, and placed in a 37°C cell culture incubator for 12 hours; 2) WELV infection of Vero cells: discard the cell supernatant, wash the cells 3 times with PBS, add 400 μL of WELV virus solution diluted in DMEM to each well, MOI=10, tap to mix, and incubate in a 37°C cell culture incubator for 1.5 hours; 3) Ribavirin and favipiravir intervention: discard the supernatant, wash with PBS three times, replace with 400 μL of ribavirin and favipiravir DMEM + 2% FBS culture medium, incubate in a 37°C cell culture incubator for 48 hours, drug concentration design: design 8 concentrations, the concentrations are 100.00µM, 50.00µM, 25.00µM, 12.50µM, 6.25µM, 3.12µM, 1.56µM, 0.78µM respectively; 4) Extraction of viral RNA from cells and RT-qPCR detection: Collect cell supernatant, wash cells 3 times with PBS, collect cells, use RNAprep Pure cultured cell / bacterial total RNA extraction kit and viral RNA extraction kit to extract viral RNA. The specific steps are carried out according to the manufacturer's instructions, and RT-qPCR is used to detect the virus level.

[0028] Table 1. Reaction system

[0029] Table 2. Reaction procedures

[0030] Table 3. Reaction system

[0031] Table 4. Reaction procedures

[0032] RT-qPCR Primers: WELV-CS2-LEFT:5'-AACCGATGGGAAAGTGACTG-3' (SEQ ID NO.1); WELV-CS2-RIGHT: 5'-GAACTTGAGGGCGTTGACAT-3' (SEQ ID NO.2); WELV-CS2-Probe:5'-FAM-ACCCTGTGCTCTGAAAGCAT-MGB-3' (SEQ ID NO.3); Vero-GAPDH-F: 5'-GGTGGTCCTCTGACTTCAACA-3' (SEQ ID NO.4); Vero-GAPDH-R: 5'-GTTGCTGTAGCCAAATTCGTTGT-3' (SEQ ID NO. 5).

[0033] 5) Results: Test results: The relative quantitative evaluation of the virus inhibition rate by measuring the intracellular virus level through real-time quantitative polymerase chain reaction (RT-qPCR) can reflect the inhibitory effect of amide-containing ribavirin and favipiravir on WELV. Figure 1 and Figure 2 It can be seen that ribavirin and favipiravir have a significant inhibitory effect on WELV in a dose-dependent manner, and the IC 50 15.41 μM and 9.6 μM respectively.

[0034] 1.2 Effects of ribavirin and favipiravir on Vero cell proliferation determined by CCK-8 cell proliferation-toxicity assay kit 1) African monkey kidney cells (Vero) were evenly plated in a 96-well plate, with 10,000 cells per well, cultured in DMEM + 2% FBS medium, and placed in a 37°C cell culture incubator for 12 hours; 2) Discard the supernatant, wash with PBS three times, replace the medium with DMEM + 2% FBS containing ribavirin and favipiravir, and incubate in a 37°C cell culture incubator for 48 hours. Drug concentration design: 8 concentrations were designed, and the concentrations were 100.00µM, 50.00µM, 25.00µM, 12.50µM, 6.25µM, 3.12µM, 1.56µM, and 0.78µM respectively; 3) The effects of ribavirin and favipiravir on cell proliferation were detected using a CCK-8 cell proliferation-toxicity assay kit according to the manufacturer's instructions.

[0035] result, Figure 3 and Figure 4 Ribavirin and favipiravir themselves have no effect on cell proliferation and have the advantages of high efficiency and low toxicity.

[0036] 1.3 Determination of the inhibitory effect of amide-free remdesivir, monaprevir, and sofosbuvir on WELV replication in vitro by real-time quantitative polymerase chain reaction (RT-qPCR) 1) African monkey kidney cells (Vero) were evenly plated in a 24-well plate, with 100,000 cells per well, cultured in DMEM + 2% FBS medium, and placed in a 37°C cell culture incubator for 12 hours; 2) Discard the cell supernatant, wash the cells 3 times with PBS, add 400 μL of WELV virus solution diluted in DMEM to each well, MOI=10, tap to mix, and incubate in a 37°C cell culture incubator for 1.5 hours; 3) Discard the supernatant, wash with PBS three times, replace with 400μL of DMEM+2%FBS medium containing remdesivir, monapravir and sofosbuvir, and incubate in a 37℃ cell culture incubator for 24h, 48h and 72h. Drug concentration design: Design 2 concentrations, which are 100.00µM and 50.00µM for sofosbuvir; 50.00µM and 25.00µM for remdesivir and monapravir; 4) Collect the cell supernatant, collect the supernatant by centrifugation, and use a viral RNA extraction kit to extract viral RNA. The specific steps are carried out according to the manufacturer's instructions. Use RT-qPCR to detect the virus level. The specific method is the same as Table 3 and Table 4.

[0037] The results showed that sofosbuvir ( Figure 5-Figure 7 )、Remdesivir( Figure 8-Figure 10 )、Monapivir( Figure 11-13 ) had no inhibitory effect on WELV 24 hours, 48 ​​hours, and 72 hours after administration.

[0038] Figure 5The average viral loads of WELV in the cell supernatant after 24 hours of administration of 100.00µM and 50.00µM sofosbuvir were 10 7.55 copies / mL and 10 7.54 copies / mL, compared with the viral load of 10 in the Vehicle group 7.52 There was no significant difference in the number of copies / mL.

[0039] Figure 6 The average viral loads of WELV in the cell supernatant were 10.00 and 50.00 μM respectively after 48 hours of administration of 100.00 μM and 50.00 μM sofosbuvir. 8.97 copies / mL and 10 8.94 copies / mL, compared with the viral load of 10 in the Vehicle group 9.04 There was no significant difference in the number of copies / mL.

[0040] Figure 7 The average viral loads of WELV in the cell supernatant after 72 hours of administration of 100.00µM and 50.00µM sofosbuvir were 10 10.53 copies / mL and 10 10.49 copies / mL, compared with the viral load of 10 in the Vehicle group 10.48 There was no significant difference in the number of copies / mL.

[0041] Figure 8 The average viral load of WELV in the cell supernatant was 10 7.63 copies / mL and 10 7.64 copies / mL, compared with the viral load of 10 in the Vehicle group 7.55 There was no significant difference in the number of copies / mL.

[0042] Fig. 9 The average viral load of WELV in the cell supernatant was 10 9.74 copies / mL and 10 9.78 copies / mL, compared with the viral load of 10 in the Vehicle group 9.79 There was no significant difference in the number of copies / mL.

[0043] Fig.10 The average viral load of WELV in the cell supernatant after 72 hours of administration of 50.00µM and 25.00µM remdesivir was 10 10.55 copies / mL and 1010.61 copies / mL, compared with the viral load of 10 in the Vehicle group 10.68 There was no significant difference in the number of copies / mL.

[0044] Fig.11 The average viral loads of WELV in the cell supernatant after 24 hours of administration of 50.00µM and 25.00µM monaprevir were 10 7.60 copies / mL and 10 7.60 copies / mL, compared with the viral load of 10 in the Vehicle group 7.55 There was no significant difference in the number of copies / mL.

[0045] Fig.12 The average viral loads of WELV in the cell supernatant were 10 9.76 copies / mL and 10 9.76 copies / mL, compared with the viral load of 10 in the Vehicle group 9.79 There was no significant difference in the number of copies / mL.

[0046] Fig.13 The average viral loads of WELV in the cell supernatant after 72 hours of administration of 50.00µM and 25.00µM monaprevir were 10 10.62 copies / mL and 10 10.57 copies / mL, compared with the viral load of 10 in the Vehicle group 10.68 There was no significant difference in the number of copies / mL. Example 2. Protective effects of ribavirin and favipiravir on WELV-infected mice

[0047] Experimental method: Six-week-old C57 BL / 6J mice were divided into 9 groups, 7 mice in each group, and infected with WELV (10 4 FFU / 100μL / mouse), and were administered by gavage on the 1st and 2nd day after infection, respectively. The dosing scheme was that each group of mice was given ribavirin 100mg / kg / day and 25mg / kg / day, favipiravir 300 mg / kg / day and 75mg / kg / day, and the positive control group (Vehicle group, the solvent was PBS), and then each group of animals was gavaged once a day for 5 days. The behavioral changes and weight changes of mice were observed every day; on the 5th day after infection, 4 mice were randomly selected for dissection, and the mouse serum, liver, spleen and lung were taken for viral load detection.

[0048] 2.1 Determination of viral load in mouse serum by real-time quantitative polymerase chain reaction (RT-qPCR): Viral RNA was extracted using a viral RNA extraction kit. The specific steps were carried out according to the manufacturer's instructions. The specific steps are the same as Table 3 and Table 4.

[0049] 2.2 Determination of viral load in mouse liver, spleen and lung by immunoplaque assay: 1) African monkey kidney cells (Vero) were evenly plated in a 24-well plate, with 100,000 cells per well, cultured in DMEM + 2% FBS medium, and placed in a 37°C cell culture incubator for 12 hours; 2) Take 10-15 mg of mouse tissue and homogenize it in DMEM to prepare a tissue suspension. After centrifugation, take the supernatant, dilute the supernatant down to 3 concentrations in a 10-fold dilution gradient and inoculate it into Vero cells. Tap to mix and incubate in a 37°C cell culture incubator for 1.5 hours. After 1.5 hours, discard the supernatant and add 1 mL of PBS to wash 3 times. Cover with culture medium containing 1.25% methylcellulose and culture at 37°C for 96 hours. 3) Add 1 mL of 3.7% formaldehyde to each well and shake horizontally for 30 min; discard the formaldehyde and culture medium mixture, add 1 mL of 3.7% formaldehyde to each well, shake well and aspirate, repeat 2-3 times; after cleaning the methylcellulose, add 1 mL of 3.7% formaldehyde to each well and shake gently on a horizontal shaker for 30 min to fix the cells; wash 5 times with 0.05% PBST (phosphate buffered saline containing Tween 20);

[0050] 4) Add 200 μL of PBS containing 0.3% Triton-X100 and 5% skim milk to each well for blocking and permeabilization, and shake gently on a horizontal shaker for 1.5 h; discard the blocking and permeabilization solution and wash 5 times with 0.05% PBST; 5) Add 200 μL of anti-WELV NP rabbit polyclonal antibody (1:1000) and incubate overnight at 4°C, then wash five times with 0.05% PBST; 6) Add 200 μL goat anti-rabbit IgG-HRP (horseradish peroxidase) antibody (1:1000) and incubate at room temperature for 1.5 h, then wash five times with 0.05% PBST; 7) Add 200 μL DAB colorimetric solution and color for 3-10 min. Discard the colorimetric solution; wash three times with 0.05% PBST, air-dry the plate, and count the spots.

[0051] Test results: Results Figure 14-Figure 29 , Fig.14 and Fig.15The survival curves of WELV-infected mice treated with ribavirin 100 mg / kg / day on the 1st and 2nd day after infection, respectively. Fig.16 and Fig.17 The survival curves of WELV-infected mice treated with 300 mg / kg / day of favipiravir starting on the first and second days after infection are shown. From the results, it can be seen that the survival rate of mice treated with higher doses of ribavirin and favipiravir starting on the first and second days after infection was 100%, and all mice in the control group died.

[0052] Fig.18 and Fig.19 Survival curves of WELV-infected mice treated with ribavirin 25 mg / kg / day starting on day 1 and day 2 after infection, respectively. Fig. 20 and 21 The following are the survival curves of WELV-infected mice treated with 75 mg / kg / day of favipiravir starting on the first and second days after infection, respectively. From the results, it can be seen that the survival rate of mice treated with lower doses of ribavirin and favipiravir starting on the first day after infection was 100%, while under the condition of lower doses of ribavirin and favipiravir starting on the second day of infection, ribavirin and favipiravir can still provide most of the protection, with mouse survival rates of 85.71% and 71.42%, respectively, and all mice in the control group died.

[0053] Figure 22-Figure 25 To quantitatively evaluate the inhibitory effect of low-dose ribavirin on the virus, the viral load in mouse serum, liver, spleen and lung was measured by RT-qPCR and immunoplaque assay. Figure 26-29 The viral load in mouse serum, liver, spleen and lung was measured by RT-qPCR and immunoplaque assay to quantitatively evaluate the inhibitory effect of low-dose favipiravir on the virus. The specific results are as follows.

[0054] Fig. 22 The viral load in the serum of mice in the control group was 10 8.17 Compared with the control group, the viral load in the serum of WELV-infected mice treated with ribavirin 25 mg / kg / day was 10 copies / mL on the first and second days after infection, respectively. 3.96 copies / mL and 10 4.76 copies / mL, and the viral load in serum decreased significantly (P < 0.001).

[0055] Fig.23 Compared with the control group, the liver viral load of mice was 10 7.56Compared with the control group, the viral load in the liver of WELV-infected mice treated with ribavirin 25 mg / kg / day was 10 FFU / mg on the first and second days after infection, respectively. 4.17 FFU / mg and 10 3.88 FFU / mg, and the viral load in the liver decreased significantly (P < 0.001).

[0056] Fig.24 Compared with the control group, the spleen viral load of mice was 10 6.94 Compared with the control group, the viral load in the spleen of WELV-infected mice treated with ribavirin 25 mg / kg / day was 10 FFU / mg on the first and second days after infection, respectively. 3.90 FFU / mg and 10 3.79 FFU / mg, and the viral load in the spleen decreased significantly (P < 0.001).

[0057] Fig.25 Compared with the control group, the lung viral load of mice was 10 6.35 Compared with the control group, the viral load in the lungs of WELV-infected mice treated with ribavirin 25 mg / kg / day was 10 FFU / mg on the first and second days after infection, respectively. 3.63 FFU / mg and 10 3.30 FFU / mg, and the viral load in the lungs decreased significantly (P < 0.001).

[0058] Fig.26 The viral load in the serum of mice in the control group was 10 8.17 Compared with the serum viral load of WELV-infected mice treated with favipiravir 75 mg / kg / day on days 1 and 2 after infection, the viral load was 10 4.41 copies / mL and 10 5.74 copies / mL, and the viral load in serum decreased significantly (P < 0.001).

[0059] Fig. 27 Compared with the control group, the liver viral load of mice was 10 7.35 Compared with the control group, the viral load in the liver of WELV-infected mice treated with favipiravir 75 mg / kg / day was 10 FFU / mg on the first and second days after infection, respectively. 4.60 FFU / mg and 10 5.05 FFU / mg, and the viral load in the liver decreased significantly (P < 0.001).

[0060] Fig.28 Compared with the control group, the spleen viral load of mice was 10 6.95Compared with the control group, the viral load in the spleen of WELV-infected mice treated with favipiravir 75 mg / kg / day was 10 FFU / mg on the first and second days after infection, respectively. 4.14 FFU / mg and 10 4.59 FFU / mg, and the viral load in the spleen decreased significantly (P < 0.001).

[0061] Fig.29 Compared with the control group, the lung viral load of mice was 10 6.45 Compared with the control group, the viral load in the lungs of WELV-infected mice treated with favipiravir 75 mg / kg / day was 10 FFU / mg on the first and second days after infection, respectively. 3.53 FFU / mg and 10 4.00 FFU / mg, and the viral load in the lungs decreased significantly (P < 0.001).

[0062] Compared with the positive control group, administration of ribavirin 25 mg / kg / day or favipiravir 75 mg / kg / day can significantly reduce the viral load in the serum, liver, spleen and lungs of mice; the above results show that ribavirin and favipiravir have a significant inhibitory effect on WELV in mice.

Claims

1. Use of non-ester nucleoside analogs or pharmaceutically acceptable salts thereof or compositions containing the same in the preparation of drugs for treating wetland viral diseases.

2. The use according to claim 1, characterized in that: The non-ester type refers to the type that does not contain a propionate group or a phosphate group.

3. The use according to claim 2, characterized in that: The non-ester nucleoside analog is a nucleoside analog containing an amide group.

4. The use according to claim 3, characterized in that: The amide-containing nucleoside analog is a nucleoside analog having formula (I): （I）。 5. The use according to claim 4, characterized in that The nucleoside analogue of formula (I) is prepared at a dosage of 25-125 mg / kg body weight.

6. The use according to claim 5, characterized in that The nucleoside analogue of formula (I) is prepared at a dosage of 100 mg / kg body weight.

7. The use according to claim 3, characterized in that: The amide-containing nucleoside analog is a nucleoside analog having formula (II): (II)。 8. The use according to claim 7, characterized in that The nucleoside analogue of formula (II) is prepared at a dosage of 75-375 mg / kg body weight.

9. The use according to claim 8, characterized in that The nucleoside analogue of formula (II) is prepared at a dose of 300 mg / kg body weight.

10. The use according to any one of claims 1 to 9, characterized in that: The drug for treating wetland viral diseases also includes a pharmaceutically acceptable carrier, and the drug is prepared in the form of tablets, capsules, solutions, emulsions, suspensions, injections, lyophilized agents, sprays, and aerosols.

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