Application of bufalin in preparing products against IHNV and IPNV

Abstract

The present invention discloses the use of bufalin in the preparation of products against IHNV and IPNV. The present invention provides the use of bufalin or its derivatives or its pharmaceutically acceptable salts or substances with bufalin or its derivatives or its pharmaceutically acceptable salts as the active ingredient in the preparation of products for anti-fish viruses; the fish viruses are infectious hematopoietic necrosis virus and / or infectious pancreatic necrosis virus. In order to screen candidate antiviral drugs against IHNV and IPNV, 1,483 traditional Chinese medicine compounds were screened from a traditional Chinese medicine monomer library. The results show that bufalin has antiviral activity against IHNV and IPNV both in vitro and in vivo, and is a potential candidate drug for anti-IHNV and IPNV infections.

Description

Technical Field

[0001] The present invention relates to the field of aquaculture, and particularly to the application of bufalin in the preparation of products against IHNV and IPNV. Background Art

[0002] Infectious hematopoietic necrosis (IHN) and infectious pancreatic necrosis (IPN) are the most common viral infectious diseases that seriously endanger the health of salmonids and are currently the two main diseases causing significant economic losses to the salmonid industry worldwide. In the 1950s, IHN was first reported in red salmon (Oncorhynchus nerka) farms in Washington and Oregon (Ammayappan et al., 2010). In the early 1980s, with the trade of fry and adult fish, IHN gradually spread to many countries in the world, such as Japan (Nishizawa et al., 2006), Iran (Ahmadivand et al., 2017), Canada (Foreman et al., 2015), South Korea (Kim et al., 2016), Russia (Rudakova et al., 2007), the Netherlands (Haenen et al., 2016) and China (Xu et al., 2019). Depending on the species and size of the fish, IHN outbreaks can result in mortality rates of over 80%, and even 100% in fry (Breyta et al., 2013; Dixon et al., 2016). Therefore, IHN is defined as a notifiable animal disease by the World Organization for Animal Health and many trading countries (Dixon et al., 2016). The pathogen of IHN is infectious hematopoietic necrosis virus (IHNV), which belongs to the family Rhabdoviridae and the genus Salmonid Novirhabdovirus (Hernandez et al., 2021). IHNV is a negative-sense single-stranded RNA virus with a genomic structure of 3'-N-P-M-G-NV-L-5', encoding a total of 5 structural proteins and 1 non-structural protein. The N gene encodes the nucleocapsid protein, the P gene encodes the phosphoprotein, the M gene encodes the matrix protein, the G gene encodes the glycoprotein, the L gene encodes the large polymerase, and the NV gene encodes the non-structural protein (Zhao et al., 2019). Currently, phylogenetic analysis shows that IHNV has evolved into 5 genotypes: U, M, L, E, and J (Xu et al., 2019). Although IHNV has caused huge economic losses to the salmonid aquaculture industry, there is only one commercial vaccine against IHNV globally, which is a DNA vaccine approved by Canada in 2005 (Alonso and Leong, 2013).This vaccine poses a risk of integrating into the host genome. Therefore, it is necessary to study more effective vaccines and antiviral drugs to prevent IHNV infection.

[0003] IPN is caused by Infectious pancreatic necrosis virus (IPNV), which is a member of the Birnaviridae family and the Aquabirnavirus genus (Gomez-Casado et al., 2011). The entire genome of IPNV is a double-stranded RNA in two segments, encoding five viral proteins, VP1, VP2, VP3, VP4, and VP5 (Ji et al., 2017). IPN was first reported in brook trout (Salvelinus fontinalis) farms in North America in the 1950s and was isolated in 1960 (Wolf et al., 1960; Wood et al., 1955). Subsequently, it has been reported in France (Wolf and Quimby, 1971), Norway (Hastein and Krogsrud, 1976; Hernandez et al., 2021), Japan (Kimura et al., 1991), Scotland (Ball et al., 1971; Benkaroun et al., 2021), Mexico (Cesar et al., 2002; Salgado-Miranda et al., 2020), the Netherlands (Haenen et al., 2016), and China (Haenen et al., 2016). The outbreak of IPN usually results in a mortality rate of 80-90% of fry, causing huge economic losses to the aquaculture industry (Bang and Kristoffersen, 2015; Dopazo, 2020). Although there are commercial vaccines against IPNV, this disease still poses major problems to the global salmon and trout aquaculture industries (Cuesta et al., 2010), and more effective antiviral drugs or vaccines need to be developed to protect fish from IPNV infection. Summary of the Invention

[0004] The object of the present invention is to provide the application of Bufalin in the preparation of products against IHNV and IPNV.

[0005] In a first aspect, the present invention claims the application of Bufalin or its derivatives or its pharmaceutically acceptable salts or substances containing Bufalin or its derivatives or its pharmaceutically acceptable salts as active ingredients in the preparation of products for anti-fish viruses; the fish viruses are Infectious hematopoietic necrosis virus and / or Infectious pancreatic necrosis virus.

[0006] In a second aspect, the present invention claims the use of Bufalin or its derivatives or its pharmaceutically acceptable salts, or a substance containing Bufalin or its derivatives or its pharmaceutically acceptable salts as an active ingredient, in the preparation of a product for preventing and / or treating diseases caused by fish virus infections; the fish virus is infectious hematopoietic necrosis virus and / or infectious pancreatic necrosis virus.

[0007] Furthermore, the disease may be infectious hematopoietic necrosis and / or infectious pancreatic necrosis.

[0008] In a third aspect, the present invention claims the use of Bufalin or its derivatives or its pharmaceutically acceptable salts, or a substance containing Bufalin or its derivatives or its pharmaceutically acceptable salts as an active ingredient, in the preparation of a product for inhibiting the attachment of infectious hematopoietic necrosis virus to the cell surface.

[0009] In a fourth aspect, the present invention claims the use of Bufalin or its derivatives or its pharmaceutically acceptable salts, or a substance containing Bufalin or its derivatives or its pharmaceutically acceptable salts as an active ingredient, in the preparation of a product for inhibiting the RNA replication of infectious hematopoietic necrosis virus.

[0010] The inhibition of the RNA replication of infectious hematopoietic necrosis virus can be specifically manifested as blocking the synthesis of infectious hematopoietic necrosis virus vRNA, mRNA, and / or cRNA.

[0011] In a fifth aspect, the present invention claims the use of Bufalin or its derivatives or its pharmaceutically acceptable salts, or a substance containing Bufalin or its derivatives or its pharmaceutically acceptable salts as an active ingredient, in the preparation of a product for inhibiting the internalization of infectious pancreatic necrosis virus on the cell surface.

[0012] In a sixth aspect, the present invention claims the use of Bufalin or its derivatives or its pharmaceutically acceptable salts, or a substance containing Bufalin or its derivatives or its pharmaceutically acceptable salts as an active ingredient, in the preparation of a product for inhibiting the RNA replication of infectious pancreatic necrosis virus.

[0013] The inhibition of the RNA replication of infectious pancreatic necrosis virus can be manifested as blocking the synthesis of infectious pancreatic necrosis virus vRNA and / or mRNA.

[0014] In each of the above aspects, the cell is a fish cell.

[0015] In a specific embodiment of the present invention, the cell is a carp epithelial cell (such as EPC cell) or a chum salmon embryo cell (such as CHSE-214 cell).

[0016] In the above aspects, the Bufalin is a compound shown in Formula I;

[0017]

[0018] In the above aspects, the substance with Bufalin or its derivative or its pharmaceutically acceptable salt as the active ingredient may be a traditional Chinese medicine complex containing Bufalin.

[0019] In order to screen candidate antiviral drugs against IHNV and IPNV, 1483 traditional Chinese medicine compounds were screened from a traditional Chinese medicine monomer library. The results showed that Bufalin (Formula I) has antiviral activity against both IHNV and IPNV in vitro and in vivo, and is a potential candidate drug for anti-IHNV and IPNV infections. Brief Description of the Drawings

[0020] Figure 1 It is the screening process of drugs against IHNV and IPNV and the structural formula of Bufalin. A is the screening process of drugs against IHNV and IPNV; B is the structural formula of Bufalin.

[0021] Figure 2 It is the detection of Bufalin cytotoxicity CC 50 and antiviral activity IC 50 Detection. A is the CC 50 detection result of Bufalin on EPC cells; B is the IC 50 detection result of Bufalin against IHNV on EPC cells; C is the CC 50 detection result of Bufalin on CHSE-214 cells; D is the IC 50 detection result of Bufalin against IPNV on CHSE-214 cells.

[0022] Figure 3 It is the detection of the inhibitory effect of Bufalin on the IHNV-Sn1203 strain. A is the detection of the inhibitory effect of Bufalin on the mRNA of the IHNV-Sn1203 strain; B is the detection of the inhibitory effect of Bufalin on the virus titer of the IHNV-Sn1203 strain; C is the detection of the inhibitory effect of Bufalin on the surface glycoprotein G of the IHNV-Sn1203 strain; D is the IFA detection of the inhibitory effect of Bufalin on the IHNV-Sn1203 strain.

[0023] Figure 4Detection of the inhibitory effect of Bufalin on different IHNV strains. A: Detection of the inhibitory effect of Bufalin on the mRNA of different IHNV strains; B: Detection of the inhibitory effect of Bufalin on the virus titer of different IHNV strains; C: Detection of the inhibitory effect of Bufalin on the surface glycoprotein G of different IHNV strains; D: IFA detection of the inhibitory effect of Bufalin on different IHNV strains.

[0024] Figure 5 Effect of Bufalin on different replication stages of IHNV. A: Effect of Bufalin on the attachment of IHNV to the cell surface; B: Effect of Bufalin on the internalization of IHNV (MOI = 10) on the cell surface; C: Effect of Bufalin on the internalization of IHNV (MOI = 100) on the cell surface; D: Effect of Bufalin on the vRNA replication of IHNV (MOI = 10) in cells; E: Effect of Bufalin on the mRNA replication of IHNV (MOI = 10) in cells; F: Effect of Bufalin on the cRNA replication of IHNV (MOI = 10) in cells; G: Effect of Bufalin on the vRNA replication of IHNV (MOI = 100) in cells; H: Effect of Bufalin on the mRNA replication of IHNV (MOI = 100) in cells; I: Effect of Bufalin on the cRNA replication of IHNV (MOI = 100) in cells..

[0025] Figure 6 Detection of the anti-IHNV virus effect of Bufalin in rainbow trout. A: Survival curve of rainbow trout challenged with IHNV after treatment with different doses of Bufalin; B: Detection of virus load in dead rainbow trout after IHNV challenge in different treatment groups; C: Detection of virus load in surviving rainbow trout after IHNV challenge in different treatment groups.

[0026] Figure 7 Detection of the inhibitory effect of Bufalin on IPNV-BJ2020-1 strain. A: Detection of the inhibitory effect of Bufalin on the mRNA of IPNV-BJ2020-1 strain; B: Detection of the inhibitory effect of Bufalin on the virus titer of IPNV-BJ2020-1 strain; C: Detection of the inhibitory effect of Bufalin on the structural protein VP2 of IPNV-BJ2020-1 strain; D: IFA detection of the inhibitory effect of Bufalin on IPNV-BJ2020-1 strain.

[0027] Figure 8 Detection of the inhibitory effect of Bufalin on different IPNV strains. A: Detection of the inhibitory effect of Bufalin on the mRNA of different IPNV strains; B: Detection of the inhibitory effect of Bufalin on the virus titer of different IPNV strains; C: Detection of the inhibitory effect of Bufalin on the structural protein VP2 of different IPNV strains; D: IFA detection of the inhibitory effect of Bufalin on different IPNV strains.

[0028] Figure 9 Effects of Bufalin on different replication stages of IPNV. A shows the effect of Bufalin on the attachment of IPNV to the cell surface; B shows the effect of Bufalin on the internalization of IPNV (MOI = 10) on the cell surface; C shows the effect of Bufalin on the internalization of IPNV (MOI = 100) on the cell surface; D shows the effect of Bufalin on the vRNA replication of IPNV (MOI = 10) inside the cell; E shows the effect of Bufalin on the mRNA replication of IPNV (MOI = 10) inside the cell; F shows the effect of Bufalin on the vRNA replication of IPNV (MOI = 100) inside the cell; G shows the effect of Bufalin on the mRNA replication of IPNV (MOI = 100) inside the cell.

[0029] Figure 10 Detection of the anti - IPNV virus effect of Bufalin in rainbow trout. A shows the detection of the IPNV virus load in rainbow trout after 1 day of virus infection treated with different doses of Bufalin; B shows the detection of the IPNV virus load in rainbow trout after 7 days of virus infection treated with different doses of Bufalin; C shows the detection of the IPNV virus load in rainbow trout after 14 days of virus infection treated with different doses of Bufalin. Specific implementation manners

[0030] The present invention will be further described in detail below in combination with specific implementation manners. The provided embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not constitute any limitation to the present invention in any way.

[0031] The experimental methods in the following embodiments, unless otherwise specified, are all conventional methods, carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following embodiments, unless otherwise specified, can all be obtained from commercial channels.

[0032] Example 1: Application of Bufalin in anti - IHNV and anti - IPNV

[0033] Traditional Chinese medicine is an important resource for developing antiviral drugs. In order to screen candidate antiviral drugs against IHNV and IPNV, we screened 1483 traditional Chinese medicine compounds from a traditional Chinese medicine monomer library. The results showed that Bufalin has antiviral activity against IHNV and IPNV both in vitro and in vivo, and is a potential candidate drug for anti - IHNV and IPNV infections.

[0034] I. Screening of anti - IHNV and anti - IPNV drugs

[0035] The traditional Chinese medicine monomer library (HY-L065) was purchased from MedChemExpress and the drugs were dissolved. First, the cell counting kit-8 (CCK8, B34304, Bimake, Shanghai, China) was used to screen for drugs with anti-IHNV activity in EPC cells (ATCC CRL-2872). The specific operations are as follows:

[0036] 1. Inoculate EPC cells into 96-well plates. When the cell density reaches 1*10 5 cells / well, co-incubate them with different drugs at a final concentration of 10 μM (diluted with PBS).

[0037] 2. After incubating for 6 h, under the condition of a virus infection multiplicity of infection (MOI) of 0.1, infect with the IHNV virus Sn1203 strain (referred to as the IHNV-Sn1203 strain) (Genbank accession number: KC660147.1) at 15 °C for 1 h.

[0038] 3. Then discard the virus solution and replace it with a culture medium containing the same drugs, and the drug concentration is also 10 μM, and culture for 7 d.

[0039] 4. Add 10 μl of CCK8 solution to each well, incubate at 15 °C for 2 h, and observe its antiviral activity. Under the condition of an optical density (OD) of 450 nm, use a microplate reader to detect cell viability.

[0040] 5. Use the same method to screen for drugs with anti-IPNV activity in CHSE-214 cells (CRL-1681, ATCC).

[0041] The results showed that Bufalin had obvious anti-IHNV and anti-IPNV activities. The specific screening protocol is as Figure 1 shown in A, and the structural formula of Bufalin is as Figure 1 shown in B.

[0042] II. Detection of Bufalin cytotoxicity CC 50 and antiviral activity IC 50

[0043] 1. CC 50 detection

[0044] Inoculate EPC cells into 96-well plates. When the cell density reaches 1*10 5 ​Each well was co-incubated with different final concentrations of Bufalin (diluted with PBS) at 1:0.01μM, 0.02μM, 0.05μM, 0.1μM, 0.2μM, 0.5μM, 1μM, 2μM, 5μM, 10μM and 20μM. Cells treated with 0.1% DMSO instead of drugs were used as the control group. After 6 days of culture, the cytotoxicity of Bufalin was detected using the CCK8 kit. The OD value of Bufalin-treated cells was 0. 450 The drug concentration that reduced the value to 50% of the control group cells was defined as the 50% cytotoxic concentration of Bufalin (CC 50 ).

[0045] CC 50 It is the drug concentration that causes 50% of cells to become diseased. The higher the value, the lower the toxicity to cells.

[0046] The results showed that Bufalin CC on EPC cells 50 >20μM( Figure 2 (A).

[0047] 2. IC 50 Detection

[0048] EPC cells in 96-well plates were also treated with different concentrations of Bufalin (same as step 1). After 6 hours of culture, IHNV was infected at 15°C for 1 hour at an MOI of 0.1. After 6 days of culture, the antiviral activity of Bufalin was detected using the CCK8 kit. The inhibition rate was calculated as [(Bufalin OD 450 - Virus control OD 450 ) / (control cell OD 450 - Virus control OD 450 )]×100%, and regression analysis was used to calculate the 50% inhibitory concentration (IC 50 ). In the calculation formula, "virus control" refers to cells treated with 0.1% DMSO and then added with virus; "control cells" refers to cells treated with 0.1% DMSO without adding virus.

[0049] IC 50 It refers to the drug concentration that can effectively inhibit 50% of cells from being infected with the virus. The smaller the value, the better the inhibitory effect on the virus.

[0050] The results showed that Bufalin's IC 50 The value is 0.1223 μM ( Figure 2 (middle B).

[0051] 3. Calculate SI

[0052] SI: Selectivity index, which is the ratio of CC 50 to IC 50 . The larger the value, the higher the probability of becoming a drug.

[0053] According to the CC in steps 1 and 2 50 and IC 50 , the selectivity index (SI = CC 50 / IC 50 ) of Bufalin against IHNV was calculated to be > 163.5.

[0054] 4. Use the same method to determine the cytotoxicity CC 50 of Bufalin and the antiviral activity IC 50 against IPNV virus on CHSE-214 cells.

[0055] The results showed that the CC of Bufalin on CHSE-214 cells 50 > 20 μM ( Figure 2 in C), and the IC 50 value of Bufalin against IPNV on CHSE-214 cells was 0.0169 μM ( Figure 2 in D). The selectivity index SI of Bufalin against IPNV was > 1183.4.

[0056] The above results indicate that the antiviral effects of Bufalin against IHNV and IPNV are not due to its cytotoxicity.

[0057] III. Bufalin inhibits the replication of IHNV in vitro and in vivo

[0058] 1. Bufalin can inhibit different IHNV virus strains

[0059] To further evaluate the anti-IHNV ability of Bufalin, we detected the replication ability of the IHNV-Sn1203 strain (Genbank accession number: KC660147.1) after being treated with Bufalin for 24 and 48 h. The specific operations are as follows: Seed EPC cells in a 6-well plate (cell density 2 * 10 6 / (hole), under the condition that the MOI of IHNV-Sn1203 was 0.1, after infecting at 15 °C for 1 h, it was replaced with fresh medium containing 0.5 μM Bufalin, and cells and culture supernatants were collected after culturing at 15 °C for 24 and 48 h. RNA was extracted from the cells using TRIzol reagent, and the mRNA (N-mRNA) expression level of the viral N gene was detected by RT-qPCR using the primers IHNV-N F / IHNV-N R in Table 1; the virus titer was determined by 10-fold serial dilution of the culture supernatant. Cells treated with 0.1% DMSO instead of the drug were used as the control group.

[0060] Table 1. Primers used for IHNV mRNA detection

[0061]

[0062] The RT-qPCR results showed that Bufalin significantly inhibited the replication of the IHNV-Sn1203 strain in cells. Compared with the DMSO control group, the relative RNA expression levels at 24 h and 48 h after Bufalin treatment decreased by 19 and 119 times, respectively ( Figure 3 in A). Compared with the DMSO control group, the extracellular virus titer of the IHNV-Sn1203 strain was also significantly inhibited, with the virus titer decreasing by 2.1 lg at 24 h and 4.3 lg at 48 h ( Figure 3 in B).

[0063] To detect the actual viral protein expression level of the IHNV-Sn1203 strain, we performed western blotting and indirect immunofluorescence assay (IFA). The specific operations were as follows: EPC cells were seeded in 6-well plates (cell density 2*10 6 / hole), under the condition that the MOI of IHNV-Sn1203 was 0.1, after infecting at 15 °C for 1 h, it was replaced with fresh medium containing 0.5 μM bufalin, and cells were collected after culturing at 15 °C for 24 and 48 h. At the same time, a mock treatment group (replacing the drug bufalin with 0.1% DMSO and not performing virus infection) and a virus control group (not adding bufalin but adding 0.1% DMSO and performing virus infection, that is Figure 3(The group labeled IHNV in the small figure D). In western blotting, after washing three times with PBS, cells were lysed on ice for 2 minutes with RIPA lysis buffer (89900, Thermo Fisher Scientific, Shanghai, China). After centrifugation at 2000×g for 10 minutes, the supernatant was run on an SDS-PAGE gel (M42010C, GenScript, Nanjing, Jiangsu province, China), and then transferred to a nitrocellulose membrane (66485, Pall Corporation, Beijing, China). After blocking with 5% skim milk powder at 37°C for 1 hour, the membrane was incubated with rabbit anti-IHNV-G antibody (polyclonal antibody, DOI code of the reference: https: / / doi.org / 10.1016 / j.molimm.2019.10.015) and rabbit anti-β-tubulin antibody (ab179513, Abcam, Cambridge, UK) as primary antibodies, and then incubated with HRP labeled goat anti-rabbit IgG antibody (ab6721, Abcam) as the secondary antibody. Finally, enhanced chemiluminescence (ECL) solution (34577, Thermo Fisher Scientific) was added, and imaging was performed using ChemiScope 6000 Touch (Clinx, Shanghai, China). In indirect immunofluorescence assay, after washing cells three times with PBS, cells were fixed with 4% (w / v) paraformaldehyde for 20 min, and then permeabilized with 0.5% (v / v) Triton X-100 at room temperature for 10 min. Cells were blocked with 5% skim milk powder in PBS at 37°C for 1 hour, and then washed three times with PBS. The cells were incubated with rabbit anti-IHNV-G antibody as the primary antibody and Cy3-tagged goat anti-rabbit IgG antibody (ab97075, Abcam) as the secondary antibody, and finally images were collected using a fluorescence microscope (DMi8, Lecia).

[0064] The results of western blotting showed that bufalin inhibited the expression level of the G protein of the IHNV-Sn1203 strain at both 24 h and 48 h ( Figure 3 as shown in C). The results of IFA showed that compared with the IHNV group (i.e., the control group without adding bufalin but adding 0.1% DMSO and infecting with the virus), the number of cells infected with the IHNV-Sn1203 strain in the bufalin treatment group was significantly reduced ( Figure 3 as shown in D).

[0065] The above results indicate that Bufalin can significantly inhibit the replication of IHNV-Sn1203 in EPC cells.

[0066] To prove that Bufalin can inhibit the infection of different IHNV virus strains, we evaluated the inhibitory effect of Bufalin on different IHNV strains Blk94 (Genbank accession number: DQ164100), LN-15 (Genbank accession number: MH170315.1), and QH-17 (Genbank accession number: MH170343.1). The specific operations are as described above, with the only difference being the replacement of the specific virus strains.

[0067] The RT-qPCR results showed that Bufalin significantly inhibited the intracellular virus replication of all viruses. At 48 h, compared with the DMSO control group, the relative RNA expression levels of Blk94, LN-15, and QH-17 were reduced by 157-fold, 39-fold, and 44-fold, respectively ( Figure 4 in A). Compared with the DMSO control group, the extracellular virus titers were also significantly inhibited. At 48 h, the virus titer of Blk94 decreased by 3.5 logs, LN-15 decreased by 3.9 logs, and QH-17 decreased by 3.8 logs ( Figure 4 in B). The western blotting results showed that Bufalin inhibited the expression levels of the G proteins of all three viruses at 48 h ( Figure 4 in C). The IFA results showed that compared with the IHNV group (i.e., the control group without adding bufalin but adding 0.1% DMSO and performing virus infection), the treatment with Bufalin significantly reduced the number of cells infected with all IHNV strains ( Figure 4 in D).

[0068] The above results indicate that Bufalin can significantly inhibit the infection of IHNV in EPC cells, and the inhibitory effect is against all virus strains, rather than a random phenomenon for a single virus strain.

[0069] 2. Bufalin inhibits the attachment of IHNV to the cell surface and the replication of viral RNA, but does not inhibit the internalization of the virus

[0070] The effective reproduction of IHNV is achieved through attachment, internalization, and RNA replication. To further study at which stage Bufalin acts, we detected the effects of Bufalin treatment on the attachment and internalization of IHNV on the cell surface.

[0071] Cell surface attachment was achieved by adding the IHNV virus and Bufalin to EPC cells (cell density 2*10 6 / well, at two virus MOI concentrations of 10 and 100, and a Bufalin drug concentration of 0.5 μM), incubate at 4 °C for 1 h to allow the virus to attach to the cell surface, then directly extract RNA for detection. The target gene is vRNA, and the primers are the 3 primers corresponding to vRNA in Table 1 (vRNA F, vRNA R, and vRNA tag; the primers for reverse transcription are vRNA F and vRNA R, and the primers for fluorescence quantitative PCR are vRNA tag and vRNA R). The RT-qPCR results showed that Bufalin could inhibit the attachment of IHNV to the cell surface at both high and low virus MOI. The inhibition rate was 41% at 10 MOI and 33% at 100 MOI( Figure 5 in A).

[0072] In the internalization experiment, EPC cells were incubated with IHNV virus at 4 °C for 1 h, and then with Bufalin at 15 °C for 30 min and 60 min (cell density 2*10 6 / well, at two virus MOI concentrations of 10 and 100, and a Bufalin drug concentration of 0.5 μM), and then directly extract RNA for detection. The target gene is vRNA, and the primers are the 3 primers corresponding to vRNA in Table 1 (vRNA F, vRNA R, and vRNA tag; the primers for reverse transcription are vRNA F and vRNA R, and the primers for fluorescence quantitative PCR are vRNA tag and vRNA R). The RT-qPCR results showed that Bufalin had no effect on the internalization of IHNV at either low MOI (10)( Figure 5 in B) or high MOI (100)( Figure 5 in C).

[0073] In the virus RNA replication experiment, first incubate EPC cells with IHNV virus for 1 h, culture at 15 °C for 2 h, then add Bufalin to the EPC cells and incubate for 4 h and 8 h (cell density 2*10 6 / well, at two virus MOI concentrations of 10 and 100, and a Bufalin drug concentration of 0.5 μM), and then directly extract RNA for RT-qPCR detection. On the one hand, detect the target gene vRNA, and the primers are the 3 primers corresponding to vRNA in Table 1 (vRNA F, vRNA R, and vRNA tag; the primers for reverse transcription are vRNA F and vRNA R, and the primers for fluorescence quantitative PCR are vRNA tag and vRNA R). The results showed that Bufalin significantly inhibited the expression level of vRNA. At 4 h, the expression level of vRNA decreased by 2.48-fold (MOI = 10) and 1.85-fold (MOI = 100), and at 8 h, the expression level of vRNA decreased by 4.17-fold (MOI = 10) and 2.5-fold (MOI = 100)( Figure 5On the other hand, the expression levels of mRNA and cRNA were detected simultaneously (primers are shown in Table 1) to further verify the inhibitory effect of Bufalin. The results showed that Bufalin had a significant inhibitory effect on the expression levels of both mRNA and cRNA. At 4 h, the mRNA expression levels decreased by 2.06-fold (MOI = 10) and 1.75-fold (MOI = 100), and at 8 h, they decreased by 3.5-fold (MOI = 10) and 2.85-fold (MOI = 100) ( Figure 5 in Figures E and H). At 4 h, the cRNA expression levels decreased by 1.7-fold (MOI = 10) and 1.95-fold (MOI = 100), and at 8 h, the cRNA expression levels decreased by 3.21-fold (MOI = 10) and 3.5-fold (MOI = 100) ( Figure 5 in Figures F and I). All these results indicated that Bufalin blocked the synthesis of IHNV vRNA, mRNA, and cRNA.

[0074] 3. Bufalin can inhibit IHNV infection in vivo

[0075] To evaluate the protective effect of Bufalin against IHNV infection in rainbow trout, 10 ± 2 g rainbow trout were treated with 50 μL (2 × 10 5 TCID 50 / mL) of IHNV and different doses of Bufalin. The specific operations were as follows:

[0076] The rainbow trout were randomly divided into 6 groups, with 50 fish in each group. When conducting the in vivo experiment on rainbow trout, Bufalin was dissolved in corn oil. Group a was intraperitoneally injected with 0.1 mg / kg of Bufalin and 50 μL (2 × 10 5 TCID 50 / mL) of IHNV, group b was intraperitoneally injected with 0.1 mg / kg of Bufalin and 50 μL of PBS, group c was intraperitoneally injected with 0.5 mg / kg of Bufalin and 50 μL (2 × 10 5 TCID 50 / mL) of IHNV, group d was intraperitoneally injected with 0.5 mg / kg of Bufalin and PBS, group e was intraperitoneally injected with 50 μL of corn oil and 50 μL (2 × 10 5 TCID 50 / mL) of IHNV, and group f was intraperitoneally injected with 50 μL of corn oil and 50 μL of PBS. The death of rainbow trout was continuously observed and counted.

[0077] The results showed that, compared with the virus-infected groups (groups a, c, e), the PBS group (groups b, d, f) did not cause the death of rainbow trout. Among them, the CPM of group e was 70%, while the CPM of the Bufalin treatment groups was relatively low. The CPM of group a (0.1 mg / kg Bufalin + IHNV) was 55%, significantly lower than that of group e (p = 0.0467). Although the RPS of group a was 21.4%, 0.1 mg / kg Bufalin could effectively delay the death process caused by IHNV and reduce the mortality rate to a certain extent. The CPM of group c (0.5 mg / kg Bufalin + IHNV) was 11.7% and the RPS was 81%, showing a highly significant difference from group e (p < 0.0001)( Figure 6 in A).

[0078] To further verify the protective effect of Bufalin against IHNV infection in vivo, the viral loads in the rainbow trout that died after virus challenge and those that survived after virus challenge were detected. The results showed that Bufalin treatment of rainbow trout did not reduce the IHNV loads in the liver, spleen and brain of the dead fish, but the viral load in the head kidney was significantly reduced. The viral load in the head kidney of the 0.1 mg / kg Bufalin group was 1.43 times lower than that of the corn oil mock treatment group (i.e., group e), and the 0.5 mg / kg Bufalin group was 3.00 times lower than that of the corn oil mock treatment group( Figure 6 in B). The viral loads in the surviving fish of the Bufalin treatment groups were all significantly reduced. The virus titers in the liver, spleen, head kidney and brain of the corn oil mock treatment group were 1.13 lg, 1.21 lg, 1.28 lg and 1.32 lg higher than those of the 0.1 mg / kg Bufalin treatment group, respectively, and 3.73 lg, 2.41 lg, 2.24 lg and 1.86 lg higher than those of the 0.5 mg / kg Bufalin treatment group, respectively( Figure 6 in C). The above results indicate that Bufalin has a significant inhibitory effect on IHNV infection in rainbow trout in vivo.

[0079] IV. Bufalin inhibits the replication of IPNV in vivo and in vitro

[0080] 1. Bufalin can inhibit different IPNV virus strains

[0081] To further evaluate the anti-IPNV ability of Bufalin, we detected the replication ability of IPNV-BJ2020-1 strain (Genbank accession number: MW662108.1) after being treated with Bufalin for 24 and 48 h. The specific operations differed from those in Step 3 only in that: the virus was replaced with IPNV; the primers used in RT-qPCR were the mRNA primers shown in Table 2; in western blotting, the primary antibodies were mouse anti-VP2 antibody (polyclonal antibody, DOI code of the reference: https: / / doi.org / 10.1016 / j.molimm.2019.10.015), and rabbit anti-β-tubulin antibody (ab179513, Abcam, Cambridge, UK) for incubation, and HRP-conjugated anti-mouse antibody (ab6728, Abcam) and HRP-conjugated anti-rabbit antibody (ab6721, Abcam) were used as secondary antibodies for incubation. In indirect immunofluorescence, the primary antibody was mouse anti-VP2 antibody (polyclonal antibody, DOI code of the reference: https: / / doi.org / 10.1016 / j.molimm.2019.10.015), and the secondary antibody was goat anti-Mouse Alexa Fluor 488 antibody (A11001, Invitrogen).

[0082] The RT-qPCR results showed that Bufalin significantly inhibited the replication of IPNV-BJ2020-1 strain in cells. Compared with the DMSO control group, the relative RNA expression levels at 24 h and 48 h after Bufalin treatment decreased by 100 and 6298 times, respectively ( Figure 7 in A). Compared with the DMSO control group, the extracellular virus titer of IPNV-BJ2020-1 strain was also significantly inhibited. The virus titer could not be detected at 24 h, and the virus titer decreased by 5.3 logs at 48 h ( Figure 7 in B). To detect the actual virus protein expression level of IPNV-BJ2020-1 strain, we also performed western blotting and indirect immunofluorescence assay (IFA). The western blotting results showed that Bufalin inhibited the expression level of VP2 protein of IPNV-BJ2020-1 strain at both 24 h and 48 h ( Figure 7In C). The IFA results showed that compared with the IPNV group (i.e., the control group without bufalin but with 0.1% DMSO and virus infection), the number of cells infected with the IPNV-BJ2020-1 strain in the Bufalin treatment group was significantly reduced ( Figure 7 In D). The above results indicate that Bufalin can significantly inhibit the replication of IPNV-BJ2020-1 in CHSE-214 cells.

[0083] To prove that Bufalin can inhibit the infection of different IPNV virus strains, we evaluated the inhibitory effect of Bufalin on different IPNV strains ChRtm213 (Genbank accession number: KX234591.1), GS2020-2 (Genbank accession number: MW662092.1), and LN2018-1 (Genbank accession number: MW662095.1). The specific operation is as described above, with the only difference being the replacement of the specific virus strain.

[0084] The RT-qPCR results showed that Bufalin significantly inhibited the intracellular virus replication of all viruses. At 48 h, compared with the DMSO control group, the relative RNA expression levels of ChRtm213, GS2020-2, and LN2018-1 were reduced by 2120-fold, 171-fold, and 99-fold, respectively ( Figure 8 In A). Compared with the DMSO control group, the extracellular virus titer was also significantly inhibited. At 48 h, the virus titer of ChRtm213 was reduced by 4.35 lg, GS2020-2 was reduced by 5.21 lg, and LN2018-1 was reduced by 5.38 lg ( Figure 8 The western blotting results showed that Bufalin inhibited the expression levels of VP2 proteins of all three viruses at 48 h ( Figure 8 In C). The IFA results showed that compared with the IPNV group (i.e., the control group without bufalin but with 0.1% DMSO and virus infection), Bufalin treatment significantly reduced the number of cells infected with all IPNV strains ( Figure 8 In D).

[0085] The above results indicate that Bufalin can significantly inhibit the infection of IPNV to CHSE-214 cells, and the inhibitory effect is against all strains, rather than a random phenomenon for a single strain.

[0086] 2. Bufalin inhibits the internalization of IPNV on the cell surface and the replication of viral RNA, but does not inhibit the attachment of the virus

[0087] To further investigate at which stage Bufalin acts, we detected the effects of Bufalin treatment on the attachment and internalization of IPNV on the cell surface.

[0088] For cell surface attachment, IPNV virus and Bufalin were added to CHSE-214 cells and incubated at 4°C for 1 h to allow the virus to attach to the cell surface (cell density 2*10 6 / well, virus MOI = 10 and 100 at two concentrations, Bufalin drug concentration 0.5 μM), and then RNA was directly extracted for detection. The target gene was vRNA, and the primers are shown in Table 2. RT-qPCR results showed that Bufalin had no effect on the attachment of IPNV to the cell surface at high and low MOI of the virus ( Figure 9 in A).

[0089] Table 2. Primers used for IPNV RNA detection

[0090]

[0091] In the internalization experiment, CHSE-214 cells were incubated with IPNV virus at 4°C for 1 h, and then with Bufalin at 15°C for 15 min, 30 min, and 60 min (cell density 2*10 6 / well, virus MOI = 10 and 100 at two concentrations, Bufalin drug concentration 0.5 μM), and then RNA was directly extracted for detection. The target gene was vRNA, and the primers are shown in Table 2. RT-qPCR results showed that Bufalin significantly inhibited the internalization of IPNV at both low MOI (10) ( Figure 9 in B) and high MOI (100) ( Figure 9 in C).

[0092] In the virus RNA replication experiment, CHSE-214 cells were first incubated with IPNV virus for 1 h, cultured at 15°C for 2 h, and then Bufalin was added to CHSE-214 cells and incubated for 4 h and 8 h, and then RNA was directly extracted for RT-qPCR detection to evaluate the effect of Bufalin on virus replication. On the one hand, we first detected the expression level of viral vRNA (primers are shown in Table 2), and the results showed that Bufalin significantly inhibited the expression level of vRNA. At 4 h, the expression level of vRNA decreased by 9.71-fold (MOI = 10) and 9.09-fold (MOI = 100), and at 8 h, the expression level of vRNA decreased by 12.17-fold (MOI = 10) and 14.07-fold (MOI = 100) ( Figure 9On the other hand, the expression levels of mRNA were simultaneously detected (primers are shown in Table 2) to further verify the inhibitory effect of Bufalin. The results showed that Bufalin also had a significant inhibitory effect on the mRNA expression levels. At 4 h, the mRNA expression levels decreased by 6.53-fold (MOI = 10) and 2.35-fold (MOI = 100), respectively, and at 8 h, they decreased by 27.78-fold (MOI = 10) and 5.43-fold (MOI = 100) ( Figure 9 in E and G). All these results indicated that Bufalin blocked the synthesis of IPNV viral vRNA and mRNA.

[0093] 3. Bufalin can inhibit IPNV infection in vivo

[0094] To evaluate the protective effect of Bufalin against IPNV infection in rainbow trout, 50 μL (1×10 6 TCID 50 / mL) of IPNV and different doses of Bufalin were used to treat 5±1 g rainbow trout. The specific operations were as follows: Rainbow trout were intraperitoneally injected with IPNV and simultaneously injected with 0.1 mg / kg or 0.5 mg / kg Bufalin (drug / fish weight). Meanwhile, a control group was set up with 50 μL of corn oil replacing Bufalin. At 1, 7, and 14 days after IPNV infection, fish tissues were collected and used to measure the viral load. 1 g of tissue was ground with 500 μl of PBS, centrifuged at 4000 rpm for 10 minutes at 4 °C, and sterilized using a 0.22 μm sterile filter. The filtered tissue fluid was serially diluted 10-fold to measure the virus titer. Three replicates were set up for each treatment group, and the tissues of 5 fish were taken and ground together in each replicate.

[0095] The results showed that, compared with the control group, at 1 day post IPNV infection, 0.1 mg / kg Bufalin significantly reduced the virus titer of IPNV in the spleen (by 0.7 lg), and 0.5 mg / kg Bufalin significantly reduced the virus titer of IPNV in the liver and spleen (by 0.8 lg in the liver and 1.1 lg in the spleen). At 7 days post IPNV infection, both 0.1 mg / kg and 0.5 mg / kg Bufalin significantly reduced the virus titer of IPNV in the liver, spleen and head kidney. Among them, 0.1 mg / kg Bufalin reduced it by 0.99 lg in the liver, 1.96 lg in the spleen and 1.65 lg in the head kidney; 0.5 mg / kg Bufalin reduced it by 2.12 lg in the liver, 2.87 lg in the spleen and 2.16 lg in the head kidney. At 14 days post IPNV infection, both 0.1 mg / kg and 0.5 mg / kg Bufalin significantly reduced the virus titer of IPNV in the liver, spleen and head kidney. Among them, 0.1 mg / kg Bufalin reduced it by 0.47 lg in the liver, 0.71 lg in the spleen and 0.68 lg in the head kidney; 0.5 mg / kg Bufalin reduced it by 1.07 lg in the liver, 1.89 lg in the spleen and 1.66 lg in the head kidney. As Figure 10 shown.

[0096] The present invention has been described in detail above. For those skilled in the art, without departing from the gist and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any modification, use or improvement of the present invention, including those that depart from the scope disclosed in this application but are made by using conventional techniques known in the art. Some basic features can be applied according to the scope of the appended claims below.

Claims

1. Application of bufalin or its pharmaceutically acceptable salt or a substance containing bufalin or its pharmaceutically acceptable salt as an active ingredient in the preparation of a product for anti-fish virus; The fish virus is infectious hematopoietic necrosis virus and / or infectious pancreatic necrosis virus; The bufalin is the compound shown in Formula I; 2. Application of bufalin or its pharmaceutically acceptable salt or a substance containing bufalin or its pharmaceutically acceptable salt as an active ingredient in the preparation of a product for preventing and / or treating diseases caused by fish virus infection; The fish virus is infectious hematopoietic necrosis virus and / or infectious pancreatic necrosis virus; The bufalin is the compound shown in Formula I; 3. The application according to claim 2, characterized in that: The diseases are infectious hematopoietic necrosis and / or infectious pancreatic necrosis.

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