CN116602970A - Application of Bufalin in preparation of IHNV and IPNV resistant products - Google Patents

Abstract

The invention discloses the application of Bufalin in preparing anti-IHNV and IPNV products. The present invention provides the application of Bufalin or its derivative or its pharmaceutically acceptable salt or the substance with Bufalin or its derivative or its pharmaceutically acceptable salt as an active ingredient in the preparation of anti-fish virus products ; The fish virus is infectious hematopoietic necrosis virus and/or infectious pancreatic necrosis virus. In order to screen out candidate antiviral drugs against IHNV and IPNV, the present invention screens 1483 Chinese medicine compounds from a Chinese medicine monomer library. The results showed that Bufalin had antiviral activity against IHNV and IPNV both in vitro and in vivo, and was a potential drug candidate against IHNV and IPNV infection.

Description

Application of Bufalin in the preparation of anti-IHNV and IPNV products

technical field

The invention relates to the field of aquaculture, in particular to the application of Bufalin in the preparation of anti-IHNV and IPNV products.

Background technique

Infectious hematopoietic necrosis (IHN) and infectious pancreatic necrosis (Infectious pancreatic necrosis, IPN) are the most common viral infectious diseases that seriously endanger the health of salmon and trout. Two of the most important diseases are responsible for significant economic losses in the salmon and trout industry. IHN was first reported in sockeye salmon (Oncorhynchus nerka) farms in Washington and Oregon in the 1950s (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 fish, outbreaks of IHN can result in over 80% mortality and even 100% mortality 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 Rhabdoviridae virus family and SalmonidNovirhabdovirus virus genus (Hernandez et al., 2021). IHNV is a negative-sense single-stranded RNA virus with a genome structure of 3'-N-P-M-G-NV-L-5', which encodes 5 structural proteins and 1 non-structural protein. The N gene encodes the nucleocapsid protein, and the P gene encodes Phosphoproteins, M genes encode matrix proteins, G genes encode glycoproteins, L genes encode large polymerases, and NV genes encode nonstructural proteins (Zhao et al., 2019). The current phylogenetic analysis shows that IHNV has evolved into five genotypes of U, M, L, E and J (Xu et al., 2019). Although IHNV has caused huge economic losses to the salmon and trout farming industry, there is only one commercial vaccine against IHNV globally, which is a DNA vaccine approved by Canada in 2005 (Alonso and Leong, 2013). There is a risk of this vaccine being integrated into the host genome, therefore, more effective vaccines and antiviral drugs are needed to prevent IHNV infection.

IPN is caused by Infectious pancreatic necrosis virus (IPNV), a member of the family Birnaviridae and the genus Aquabirnavvirus (Gomez-Casado et al., 2011). The entire genome of IPNV is a double-segment double-stranded RNA encoding five viral proteins VP1, VP2, VP3, VP4, and VP5 (Ji et al., 2017). IPN was first reported in North American brook trout (Salvelinus fontinalis) farms in the 1950s and isolated in 1960 (Wolf et al., 1960; Wood et al., 1955). Subsequently, 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) have also been reported. Outbreaks of IPN usually result in 80-90% mortality of fry, causing huge economic losses to the farming industry (Bang and Kristoffersen, 2015; Dopazo, 2020). Although a commercial vaccine against IPNV is available, the disease still poses a major problem to the global salmon and trout farming industry (Cuesta et al., 2010), and the development of more effective antiviral drugs or vaccines to protect fish against Infected by IPNV.

Contents of the invention

The purpose of the present invention is to provide the application of Bufalin in the preparation of anti-IHNV and IPNV products.

In the first aspect, the present invention claims to protect Bufalin or its derivative or its pharmaceutically acceptable salt or the material with Bufalin or its derivative or its pharmaceutically acceptable salt as active ingredient in the preparation of anti-fish virus Application in products; the fish virus is infectious hematopoietic necrosis virus and/or infectious pancreatic necrosis virus.

In the second aspect, the present invention claims to protect Bufalin or its derivatives or its pharmaceutically acceptable salt or the substance with Bufalin or its derivatives or its pharmaceutically acceptable salt as the active ingredient in the preparation for prevention and/or treatment Application in products for diseases caused by fish virus infection; said fish virus is infectious hematopoietic necrosis virus and/or infectious pancreatic necrosis virus.

Further, the disease may be infectious hematopoietic necrosis and/or infectious pancreatic necrosis.

In the third aspect, the present invention claims to protect Bufalin or its derivative or its pharmaceutically acceptable salt or the substance with Bufalin or its derivative or its pharmaceutically acceptable salt as the active ingredient in the preparation for inhibiting infectious hematopoietic organ Application of products for attachment of necrotic viruses to cell surfaces.

In the fourth aspect, the present invention claims to protect Bufalin or its derivatives or its pharmaceutically acceptable salts or the substances with Bufalin or its derivatives or its pharmaceutically acceptable salts as active ingredients in the preparation for inhibiting infectious hematopoietic organ Application of products of necrotic viral RNA replication.

Said inhibition of infectious hematopoietic organ necrosis virus RNA replication can specifically be embodied as blocking the synthesis of infectious hematopoietic organ necrosis virus vRNA, mRNA and/or cRNA.

In the fifth aspect, the present invention claims to protect Bufalin or its derivatives or its pharmaceutically acceptable salts or substances with Bufalin or its derivatives or its pharmaceutically acceptable salts as active ingredients in the preparation for inhibiting infectious pancreatic Application of necrotic virus in the product of internalization at the cell surface.

In the sixth aspect, the present invention claims to protect Bufalin or its derivatives or its pharmaceutically acceptable salts or substances with Bufalin or its derivatives or its pharmaceutically acceptable salts as active ingredients in the preparation for inhibiting infectious pancreatic Application of products of necrotic viral RNA replication.

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

In the above aspects, the cells are fish cells.

In a specific embodiment of the present invention, the cells are carp epithelial cells (such as EPC cells) or chinook salmon embryo cells (such as CHSE-214 cells).

In the above aspects, the Bufalin is a compound represented by formula I;

In the above aspects, the substance with Bufalin or its derivatives or a pharmaceutically acceptable salt thereof as an active ingredient may be a traditional Chinese medicine compound containing Bufalin.

In order to screen out candidate antiviral drugs against IHNV and IPNV, the present invention screens 1483 Chinese medicine compounds from a Chinese medicine monomer library. The results show that Bufalin (formula I) has antiviral activity against IHNV and IPNV in vitro and in vivo, and is a potential drug candidate against IHNV and IPNV infection.

Description of drawings

Figure 1 is the anti-IHNV, IPNV drug screening process and the structural formula of Bufalin. A is the anti-IHNV, IPNV drug screening process; B is the structural formula of Bufalin.

Figure 2 shows the detection of Bufalin cytotoxicity CC ₅₀ and antiviral activity IC ₅₀ . A is the detection result of CC ₅₀ of Bufalin on EPC cells; B is the detection result of IC ₅₀ of Bufalin on IHNV on EPC cells; C is the detection result of CC ₅₀ of Bufalin on CHSE-214 cells; D is the detection result of Bufalin on CHSE-214 IC ₅₀ detection results for IPNV on cells.

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

Figure 4 is the detection of the inhibitory effect of Bufalin on different IHNV strains. A is detection of mRNA inhibitory effect on different IHNV strains; B is detection of inhibitory effect on virus titer of different IHNV strains; C is detection of inhibitory effect on surface glycoprotein G of different IHNV strains; D is inhibition of different IHNV strains IFA detection of effect.

Figure 5 shows the effect of Bufalin on different replication stages of IHNV. A is the effect of Bufalin on the attachment of IHNV on the cell surface; B is the effect of Bufalin on the internalization of IHNV (MOI=10) on the cell surface; C is the effect of Bufalin on the internalization of IHNV (MOI=100) on the cell surface; D is Effect of Bufalin on IHNV (MOI=10) replication of vRNA in cells; E is effect of Bufalin on replication of mRNA in cells of IHNV (MOI=10); F is effect of Bufalin on replication of cRNA in cells of IHNV (MOI=10) Effect; G is the effect of Bufalin on the replication of IHNV (MOI=100) in the cell vRNA; H is the effect of Bufalin on the replication of the mRNA in the cell of IHNV (MOI=100); I is the effect of Bufalin on the replication of IHNV (MOI=100) in the cell Effects on endogenous cRNA replication. .

Figure 6 is the detection of Bufalin's anti-IHNV virus effect in rainbow trout. A is the survival curve of rainbow trout challenged with IHNV after different doses of Bufalin treatment; B is the detection of virus load in dead rainbow trout after IHNV challenge in different treatment groups; C is the detection of viral load in surviving rainbow trout after IHNV challenge in different treatment groups.

Figure 7 shows the detection of the inhibitory effect of Bufalin on the IPNV-BJ2020-1 strain. A is the detection of the inhibitory effect on IPNV-BJ2020-1 strain mRNA; B is the detection of the inhibitory effect on the virus titer of the IPNV-BJ2020-1 strain; C is the detection of the inhibitory effect on the structural protein VP2 of the IPNV-BJ2020-1 strain; D is the IFA detection of the inhibitory effect on the IPNV-BJ2020-1 strain.

Figure 8 is the detection of the inhibitory effect of Bufalin on different IPNV strains. A is the detection of the inhibitory effect on mRNA of different IPNV strains; B is the detection of the inhibitory effect on the virus titer of different IPNV strains; C is the detection of the inhibitory effect on the structural protein VP2 of different IPNV strains; D is the inhibitory effect on different IPNV strains The IFA test.

Figure 9 shows the effect of Bufalin on different replication stages of IPNV. A is the effect of Bufalin on the attachment of IPNV on the cell surface; B is the effect of Bufalin on the internalization of IPNV (MOI=10) on the cell surface; C is the effect of Bufalin on the internalization of IPNV (MOI=100) on the cell surface; D is The effect of Bufalin on the replication of IPNV (MOI=10) in intracellular vRNA; E is the effect of Bufalin on the replication of IPNV (MOI=10) in intracellular mRNA; F is the effect of Bufalin on the replication of IPNV (MOI=100) in intracellular vRNA Effect; G is the effect of Bufalin on mRNA replication of IPNV (MOI=100) in cells.

Figure 10 is the detection of the anti-IPNV virus effect of Bufalin in rainbow trout. A is the detection of IPNV virus load in rainbow trout treated with different doses of Bufalin for 1 day after virus infection; B is the detection of IPNV virus load in rainbow trout treated with different doses of Bufalin for 7 days after virus infection; C is the detection of IPNV virus in rainbow trout after treated with different doses of Bufalin for 14 days load detection.

Detailed ways

The present invention will be further described in detail below in conjunction with specific embodiments, and the given examples are only for clarifying the present invention, not for limiting the scope of the present invention. The examples provided below can be used as a guideline for those skilled in the art to make further improvements, and are not intended to limit the present invention in any way.

The experimental methods in the following examples, unless otherwise specified, are 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 and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Embodiment 1, the application of Bufalin in anti-IHNV and IPNV

Traditional Chinese medicine is an important resource for the development of antiviral drugs. In order to screen out candidate antiviral drugs against IHNV and IPNV, we screened 1483 traditional Chinese medicine compounds from a single traditional Chinese medicine library. The results showed that Bufalin had antiviral activity against IHNV and IPNV both in vitro and in vivo, and was a potential drug candidate against IHNV and IPNV infection.

1. Screening of anti-IHNV and IPNV drugs

The Chinese medicine monomer library (HY-L065) was purchased from MedChemExpress, and the medicine had been dissolved. First, EPC cells (ATCC CRL-2872) were screened for anti-IHNV active drugs using the cellcounting kit-8 kit (CCK8, B34304, Bimake, Shanghai, China). The specific operation is as follows:

1. Inoculate EPC cells in a 96-well plate, and co-incubate with different drugs at a final concentration of 10 μM (diluted with PBS) when the cell density reaches 1*10 ⁵ cells/well.

2. After incubation for 6 hours, the virus was infected with IHNV virus Sn1203 strain (referred to as IHNV-Sn1203 strain) (Genbank accession number: KC660147.1) at 15° C. for 1 hour under the condition of virus infection concentration MOI of 0.1.

3. Then discard the virus liquid and replace it with a culture liquid containing the same drug, the drug concentration is also 10 μM, and culture for 7 days.

4. Add 10 μl CCK8 solution to each well, incubate at 15°C for 2 hours, and observe its antiviral activity. Under the condition of optical density (OD) 450nm, the cell viability was detected with a microplate reader.

5. Use the same method to screen anti-IPNV active drugs on CHSE-214 cells (CRL-1681, ATCC).

The results showed that: Bufalin has obvious anti-IHNV, IPNV activity. The specific screening scheme is shown in A in Figure 1, and the structural formula of Bufalin is shown in B in Figure 1.

2. Detection of Bufalin Cytotoxicity CC ₅₀ and Antiviral Activity IC ₅₀

1. CC ₅₀ detection

Seed EPC cells in a 96-well plate, and co-incubate with different final concentrations of Bufalin (diluted with PBS) when the cell density reaches 1*10 ⁵ cells/well, and set the drug concentration as follows: 0.01 μM, 0.02 μM, 0.05 μM , 0.1 μM, 0.2 μM, 0.5 μM, 1 μM, 2 μM, 5 μM, 10 μM and 20 μM. Cells treated with 0.1% DMSO as a control group. After culturing for 6 days, the cytotoxicity of Bufalin was detected with CCK8 kit. The 50% cytotoxic concentration (CC ₅₀ ) of Bufalin was defined as the drug concentration that reduced the OD ₄₅₀ value of Bufalin-treated cells to 50% of that of control cells.

CC ₅₀ is the concentration of the drug at which 50% of the cells are damaged, and the higher the value, the lower the toxicity to the cells.

The results showed that the CC ₅₀ of Bufalin on EPC cells was >20 μM (A in FIG. 2 ).

2. _IC50 detection

The EPC cells in the 96-well plate were also treated with different concentrations of Bufalin (same as step 1). After culturing for 6 hours, they were infected at 15° C. for 1 hour under the condition of IHNV MOI=0.1. After culturing for 6 days, the antiviral activity of Bufalin was detected with CCK8 kit. Calculate the inhibition rate [(Bufalin OD ₄₅₀ - virus control OD ₄₅₀ )/(control cell OD ₄₅₀ - virus control OD ₄₅₀ )]×100%, and use regression analysis to calculate the 50% inhibitory concentration (IC ₅₀ ) of Bufalin to IHNV. In the calculation formula, "virus control" refers to adding virus after treating cells with 0.1% DMSO; "control cell" refers to treating cells with 0.1% DMSO without adding virus.

IC ₅₀ refers to the drug concentration that can effectively inhibit 50% of the cells infected with the virus, and the smaller the value, the better the inhibitory effect on the virus.

It was found that the _IC50 value of Bufalin was 0.1223 μM (B in Figure 2).

3. Calculate SI

SI: selectivity index, which is the ratio of CC ₅₀ to IC ₅₀ , the larger the value, the higher the possibility of becoming a drug.

According to the calculation of CC ₅₀ and IC ₅₀ in steps 1 and 2, the selectivity index of Bufalin to IHNV (SI=CC ₅₀ /IC ₅₀ )>163.5.

4. Determine the CC ₅₀ of Bufalin cytotoxicity and the IC ₅₀ of anti-IPNV virus activity on CHSE-214 cells by the same method.

The result shows: the CC of Bufalin on CHSE-214 cells > ₂₀ μ M (C in Fig. 2), the _IC value of IPNV on CHSE-214 cells is 0.0169 μ M (D in Fig. 2), the selectivity of Bufalin to IPNV Index SI was >1183.4.

The above results indicated that the antiviral effect of Bufalin on IHNV and IPNV was not caused by its cytotoxicity.

3. Bufalin inhibits IHNV replication in vivo and in vitro

1. Bufalin can inhibit different IHNV strains

In order to further evaluate the anti-IHNV ability of Bufalin, we detected the replication ability of IHNV-Sn1203 strain (Genbank accession number: KC660147.1) after Bufalin treatment for 24 and 48 hours. The specific operation is as follows: inoculate EPC cells in a 6-well plate (cell density 2*10 ⁶ /well), under the condition of IHNV-Sn1203 MOI=0.1, after infection at 15°C for 1 hour, replace with fresh medium containing 0.5μM Bufalin, cells and culture supernatant were collected after cultured at 15°C for 24 and 48 hours. Wherein cells utilize TRIzol reagent to extract RNA, adopt the primer IHNV-NF/IHNV-NR in Table 1 to utilize the method for RT-qPCR to detect the mRNA (N-mRNA) expression level of virus N gene; Culture supernatant adopts 10 times gradient Dilution method to determine virus titer. Cells treated with 0.1% DMSO as a control group.

Table 1. Primers used for IHNV mRNA detection

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

In order to detect the actual IHNV-Sn1203 strain viral protein expression level, we performed western blotting and indirect immunofluorescence (IFA). The specific operation is as follows: inoculate EPC cells in a 6-well plate (cell density 2*10 ⁶ /well), under the condition of IHNV-Sn1203 MOI=0.1, after infection at 15°C for 1 hour, replace with fresh medium containing 0.5 μM bufalin, cells were collected after 24 and 48 hours of culture at 15°C. Simultaneously set the simulated treatment group (use 0.1% DMSO instead of drug bufalin and do not carry out virus infection) and virus control group (do not add bufalin but add 0.1% DMSO and carry out virus infection, that is, the group marked IHNV in small panel D in Fig. 3 ). In western blotting, after three washes with PBS, lyse with RIPA lysis buffer (89900, Thermo Fisher Scientific, Shanghai, China) for 2 minutes on ice. 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% skimmed milk powder at 37°C for 1 hour, use rabbitanti-IHNV-G antibody (polyantibody, DOI code of reference: https://doi.org/10.1016/j.molimm.2019.10.015 ), and rabbit anti-β-tubulin antibody (ab179513, Abcam, Cambridge, UK) as the primary antibody, and 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 ChemiScope 6000Touch (Clinx, Shanghai, China) was used for imaging. In indirect immunofluorescence, cells were washed with PBS three times, 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% nonfat dry milk in PBS at 37°C for 1 hour, and then washed three times with PBS. Incubation was performed with rabbitanti-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 with a fluorescence microscope (DMi8, Lecia).

The results of Western blotting showed that Bufalin inhibited the expression level of the G protein of the IHNV-Sn1203 strain at 24h and 48h (C in Figure 3). The results of IFA showed that compared with the IHNV group (that is, the control group without adding bufalin but adding 0.1% DMSO and carrying out virus infection), the number of cells infected by the IHNV-Sn1203 strain in the Bufalin treatment group was significantly reduced (D in Figure 3).

The above results indicated that Bufalin could significantly inhibit the replication of IHNV-Sn1203 in EPC cells.

In order to prove that Bufalin can inhibit the infection of different IHNV strains, we evaluated the 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: No.: MH170343.1) inhibitory effect. See the above for specific operations, the only difference is to replace specific strains.

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

The above results show that: Bufalin can significantly inhibit the infection of EPC cells by IHNV, and the inhibitory effect is on all strains, rather than a random phenomenon on a single strain.

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

IHNV infection achieves efficient virus propagation through attachment, internalization, and RNA replication. To further investigate at which stage Bufalin acts, we examined the effect of Bufalin treatment on the attachment and internalization of IHNV on the cell surface.

For cell surface attachment, add IHNV virus and Bufalin to EPC cells (cell density 2*10 ⁶ /well, virus MOI = 10 and 100 concentrations, Bufalin drug concentration 0.5 μM), and incubate at 4°C for 1 hour to allow the virus to attach to The target gene is vRNA, and the primers are the three 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 fluorescent Quantitative primers are vRNA tag and vRNA R). RT-qPCR results showed that Bufalin could inhibit IHNV from attaching to the cell surface at both high and low MOI of the virus, the inhibition rate was 41% at 10 MOI, and 33% at 100 MOI (A in Figure 5).

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

In the viral RNA replication experiment, EPC cells were first incubated with IHNV virus for 1 hour, and then cultured at 15°C for 2 hours, then Bufalin was added to the EPC cells and incubated for 4 hours and 8 hours (cell density 2*10 ⁶ /well, virus MOI=10 and 100 two Concentration, Bufalin drug concentration 0.5μM), and then directly extract RNA for RT-qPCR detection. On the one hand, target gene vRNA is detected, and the primers are 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 fluorescent quantification are vRNA tag and vRNA tag R). The results showed that Bufalin significantly inhibited the expression level of vRNA. At 4h, the expression level of vRNA was reduced by 2.48 times (MOI=10) and 1.85 times (MOI=100), and at 8h, the expression level of vRNA was reduced by 4.17 times ( MOI=10) and 2.5 times (MOI=100) (D and G in Figure 5). On the other hand, the expression levels of mRNA and cRNA were detected simultaneously (see Table 1 for primers) to further verify the inhibitory effect of Bufalin. The results showed that Bufalin had a significant inhibitory effect on both mRNA and cRNA expression levels. The mRNA expression levels decreased by 2.06 times (MOI=10) and 1.75 times (MOI=100) at 4h, and 3.5 times (MOI=10) and 2.85 times (MOI=100) at 8h (Figure 5 E and H), At 4h, the cRNA expression levels decreased by 1.7 times (MOI=10) and 1.95 times (MOI=100), respectively, and at 8h, the cRNA expression levels decreased by 3.21 times (MOI=10) and 3.5 times (MOI=100) (Fig. 5 F and I). All these results indicated that Bufalin blocked the synthesis of IHNV vRNA, mRNA and cRNA.

3. Bufalin can inhibit IHNV infection in vivo

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

Rainbow trout were randomly divided into 6 groups, 50 fish in each group. Bufalin was dissolved in corn oil when conducting rainbow trout in vivo experiments. In group a, 0.1 mg/kg of Bufalin and 50 μL (2×10 ⁵ TCID ₅₀ /mL) of IHNV were injected intraperitoneally at the same time; in group b, 0.1 mg/kg of Bufalin and 50 μL of PBS were injected intraperitoneally at the same time; in group c, 0.5 mg/kg was injected intraperitoneally at the same time Bufalin and 50μL (2×10 ⁵ TCID ₅₀ /mL) of IHNV, group d was injected intraperitoneally with 0.5mg/kg Bufalin and PBS at the same time, and group e was injected with 50μL corn oil and 50μL (2×10 ⁵ TCID ₅₀ /mL) ) of IHNV, and group f were injected intraperitoneally with 50 μL corn oil and 50 μL PBS at the same time. The rainbow trout mortality was continuously observed and counted.

The results showed that the PBS group (group b, d, f) did not cause the death of rainbow trout compared with the virus-infected group (group a, c, e). Among them, the CPM of group e was 70%, while the CPM of the Bufalin treatment group was lower, and the CPM of group a (0.1 mg/kg Bufalin+IHNV) was 55%, which was significantly lower than that of group e (p=0.0467). Although the RPS of group a was 21.4%, 0.1mg/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 (0.5mg/kg Bufalin+IHNV) was 11.7%, and the RPS was 81%, which were significantly different from group e (p<0.0001) (A in Figure 6).

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

4. Bufalin inhibits IPNV replication in vivo and in vitro

1. Bufalin can inhibit different IPNV strains

In order to further evaluate the anti-IPNV ability of Bufalin, we detected the replication ability of IPNV-BJ2020-1 strain after Bufalin treatment for 24 and 48h (Genbank accession number: MW662108.1). The only difference between the specific operation and step 3 1 is that the virus is replaced by IPNV; the primers used for RT-qPCR refer to the mRNA primers in Table 2; the primary antibody in Western blotting is mouse anti-VP2 antibody (polyantibody, reference The DOI code is: https://doi.org/10.1016/j.molimm.2019.10.015), and rabbit anti-β-tubulin antibody (ab179513, Abcam, Cambridge, UK) was used as the primary antibody for incubation, and HRP-conjugated Anti-mouse antibody (ab6728, Abcam) and HRP-conjugated anti-rabbit antibody (ab6721, Abcam) were incubated as secondary antibodies. In indirect immunofluorescence, the primary antibody is mouse anti-VP2 antibody (polyantibody, the DOI code of the reference is: https://doi.org/10.1016/j.molimm.2019.10.015), and the secondary antibody is goat anti-Mouse Alexa Fluor 488 antibody (A11001, Invitrogen).

The results of RT-qPCR showed that Bufalin significantly inhibited the replication of the IPNV-BJ2020-1 strain in cells. Compared with the DMSO control group, the relative RNA expression levels at 24h and 48h after Bufalin treatment were reduced by 100 and 6298 times, respectively (Figure 7 Middle A). Compared with the DMSO control group, the extracellular virus titer of the IPNV-BJ2020-1 strain was also significantly suppressed, the virus titer could not be detected at 24h, and the virus titer was reduced by 5.3 lg at 48h (B in Figure 7). In order to detect the actual IPNV-BJ2020-1 strain viral protein expression level, we also performed western blotting and indirect immunofluorescence (IFA). The results of western blotting showed that Bufalin inhibited the expression level of the VP2 protein of the IPNV-BJ2020-1 strain at 24h and 48h (C in Figure 7). The results of IFA showed that compared with the IPNV group (that is, the control group without adding bufalin but adding 0.1% DMSO and carrying out virus infection), the number of infected cells of the IPNV-BJ2020-1 strain in the Bufalin treatment group was significantly reduced (D in Figure 7). The above results indicated that Bufalin could significantly inhibit the replication of IPNV-BJ2020-1 in CHSE-214 cells.

In order to prove that Bufalin can inhibit the infection of different IPNV strains, we evaluated the 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) inhibitory effect. See the above for specific operations, the only difference is to replace specific strains.

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

The above results show that Bufalin can significantly inhibit the infection of IPNV on CHSE-214 cells, and the inhibitory effect is on all strains, rather than a random phenomenon on a single strain.

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

To further investigate at which stage Bufalin acts, we examined the effect of Bufalin treatment on the attachment and internalization of IPNV on the cell surface.

Cell surface attachment is to add IPNV virus and Bufalin into CHSE-214 cells, incubate at 4°C for 1 hour to make the virus attach to the cell surface (cell density 2*10 ⁶ /well, two concentrations of virus MOI=10 and 100, Bufalin drug concentration 0.5 μM), and then directly extract RNA for detection, the target gene is vRNA, and the primers are shown in Table 2. The results of RT-qPCR showed that Bufalin had no effect on the attachment of IPNV on the cell surface at high or low MOI of the virus (A in Figure 9).

Table 2. Primers used for IPNV RNA detection

In the internalization experiment, CHSE-214 cells were incubated with IPNV virus at 4°C for 1h, and then incubated with Bufalin at 15°C for 15min, 30min and 60min (cell density 2*10 ⁶ /well, virus MOI=10 and 100 two concentrations , Bufalin drug concentration 0.5 μM), and then directly extract RNA for detection, the target gene is vRNA, and the primers are shown in Table 2. T-qPCR results showed that Bufalin had a significant inhibitory effect on the internalization of IPNV at both low MOI (10) (B in Figure 9) and high MOI (100) (C in Figure 9).

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

3. Bufalin can inhibit IPNV infection in vivo

To evaluate the protective effect of Bufalin on IPNV infection in rainbow trout, 5±1g rainbow trout were treated with 50μL (1×10 ⁶ TCID ₅₀ /mL) of IPNV and different doses of Bufalin. The specific operation is as follows: intraperitoneal injection of IPNV and 0.1 mg/kg or 0.5 mg/kg Bufalin (drug/fish weight) to rainbow trout. At the same time, a control group in which 50 μL of corn oil was used instead of Bufalin was set. After 1, 7 and 14 days of IPNV infection, fish tissues were collected and used to determine viral load. One gram of tissue was ground with 500 μl PBS, centrifuged at 4000 rpm for 10 min at 4 °C, and sterilized using a 0.22 μm sterile filter. The filtered tissue fluid was serially diluted 10-fold to determine the virus titer. Three repetitions were set up for each treatment group, and the tissues of 5 fish were taken for mixing and grinding in each repetition.

The results showed that, compared with the control group, 0.1mg/kg Bufalin significantly reduced the viral titer of IPNV in the spleen (by 0.7 lg) when IPNV was infected for 1 day, and 0.5mg/kg Bufalin significantly reduced the viral titer of IPNV in the liver and spleen. Viral titers of IPNV (0.8 lg decreased in liver and 1.1 lg decreased in spleen). At 7 days after IPNV infection, both 0.1mg/kg and 0.5mg/kg Bufalin significantly reduced the virus titer of IPNV in the liver, spleen and head kidney, among which 0.1mg/kgBufalin reduced 0.99 lg in the liver and 0.99 lg in the spleen. Reduced by 1.96 lg and 1.65 lg in the head kidney; 0.5mg/kg Bufalin decreased by 2.12 lg in the liver, 2.87 lg in the spleen, and 2.16 lg in the head kidney. At 14 days after IPNV infection, both 0.1mg/kg and 0.5mg/kg Bufalin significantly reduced the virus titer of IPNV in the liver, spleen and head kidney, among which 0.1mg/kg Bufalin reduced 0.47 lg in the liver and 0.47 lg in the spleen 0.71 lg in the liver, 0.68 lg in the head kidney; 0.5mg/kg Bufalin decreased 1.07 lg in the liver, 1.89 lg in the spleen, and 1.66 lg in the head kidney. As shown in Figure 10.

The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention, and without unnecessary experiments, the present invention can be practiced in a wider range under equivalent parameters, concentrations and conditions. While specific embodiments of the invention have been shown, it should be understood that the invention can be further modified. In a word, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by using conventional techniques known in the art and departing from the disclosed scope of this application. Applications of some of the essential features are possible within the scope of the appended claims below.

Claims (10)

1. Application of Bufalin or its derivatives or pharmaceutically acceptable salts thereof or substances containing Bufalin or its derivatives or pharmaceutically acceptable salts as active ingredients in the preparation of anti-fish virus products; The fish virus is infectious hematopoietic necrosis virus and/or infectious pancreatic necrosis virus. 2. Bufalin or its derivatives or pharmaceutically acceptable salts thereof or substances with Bufalin or its derivatives or pharmaceutically acceptable salts thereof as active ingredients are used in the preparation for the prevention and/or treatment of fish virus infection Use in products that cause disease; The fish virus is infectious hematopoietic necrosis virus and/or infectious pancreatic necrosis virus. 3. The application according to claim 2, characterized in that the disease is infectious hematopoietic necrosis and/or infectious pancreatic necrosis. 4. Bufalin or its derivatives or pharmaceutically acceptable salts thereof or substances with Bufalin or its derivatives or pharmaceutically acceptable salts thereof as active ingredients are used in the preparation for inhibiting transmissible hematopoietic organ necrosis virus on the cell surface application of attached products. 5. Bufalin or its derivatives or their pharmaceutically acceptable salts or substances with Bufalin or its derivatives or their pharmaceutically acceptable salts as active ingredients are used in the preparation of products for inhibiting the RNA replication of infectious hematopoietic organ necrosis virus in the application. 6. Bufalin or its derivatives or pharmaceutically acceptable salts thereof, or substances with Bufalin or its derivatives or pharmaceutically acceptable salts thereof as active ingredients are used in the preparation for inhibiting infectious pancreatic necrosis virus on the cell surface Internalized product applications. 7. Bufalin or its derivatives or their pharmaceutically acceptable salts or substances with Bufalin or its derivatives or their pharmaceutically acceptable salts as active ingredients are used in the preparation of products for inhibiting the RNA replication of infectious pancreatic necrosis virus in the application. 8. The application according to any one of claims 1-7, characterized in that: the cells are fish cells. 9. The application according to claim 8, characterized in that: the cells are carp epithelial cells or chinook salmon embryo cells. 10. according to the application described in any one in claim 1-9, it is characterized in that: described Bufalin is the compound shown in formula I;