Application of heparan sulfate in prevention and treatment of grass carp reovirus

Abstract

The invention relates to the technical field of aquaculture, in particular to application of heparan sulfate in prevention and treatment of grass carp reovirus. The heparan sulfate is competitively combined with the grass carp reovirus, so that the virus adsorption and infection process can be effectively inhibited. In drugs, fish feed and additives, when the concentration of heparan sulfate exceeds 0.1 mu g / mL or 10mg / kg, the effect of resisting grass carp reovirus is obviously shown. Cell experiments and artificial infection challenge experiments prove that the virus infection rate can be reduced, the survival rate can be increased, and tissue damage can be reduced. As an antiviral feed additive, the heparan sulfate provided by the invention has the advantages of low cost, small dosage, high safety and the like, provides a new strategy for preventing and treating viral diseases in the aquaculture industry, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of aquaculture, and particularly to the application of heparan sulfate in preventing and treating grass carp reovirus. Background Art

[0002] As a pillar fish in China's freshwater aquaculture industry, grass carp has significant economic value, accounting for approximately 21% of the freshwater fish aquaculture output. However, a major challenge in grass carp aquaculture is the hemorrhagic disease caused by grass carp reovirus (GCRV). This disease is characterized by a fast transmission speed and extremely high lethality. After infection, it can cause extensive congestion and bleeding in various organs and tissues of grass carp, with a mortality rate of over 80%, bringing huge economic losses to China's grass carp aquaculture industry. Although grass carp hemorrhagic disease has been classified as a class II animal disease, current prevention and control measures are scarce, especially the lack of safe and effective prevention and treatment drugs and preventive vaccines. Existing prevention and control measures cannot effectively contain the spread of the disease, posing a serious threat to the healthy development of the grass carp aquaculture industry.

[0003] In view of the serious harm of grass carp hemorrhagic disease and the deficiencies of existing prevention and control measures, there is an urgent need to develop a new type of anti-GCRV drug. This drug should have the characteristics of broad-spectrum, high efficiency, safety, pollution-free, and low cost to meet the urgent needs of the current grass carp aquaculture industry. Heparan sulfate (HS), as a polysaccharide widely distributed on the surface of mammalian cells and in the extracellular matrix, its important role in regulating the binding of signaling molecules to receptors has been widely recognized. Many viruses use heparan sulfate as an adsorption receptor to initiate the infection process, but the role of heparan sulfate in GCRV infection is not clear, and there is currently no report on using heparan sulfate to prevent and treat GCRV infection. Summary of the Invention

[0004] In view of this, the present invention proposes the application of heparan sulfate in preventing and treating grass carp reovirus.

[0005] The technical solution of the present invention is realized as follows:

[0006] In the first aspect, the present invention provides the application of heparan sulfate in the preparation of a drug for preventing and treating grass carp reovirus.

[0007] In some specific embodiments, the application includes:

[0008] 1. Heparan sulfate is used as the active ingredient of the drug, and the drug also contains pharmaceutically acceptable excipients.

[0009] 2. The dosage of heparan sulfate is divided into the following two types:

[0010] A1: The dosage is at least 10 mg / kg (suitable for administration methods such as feeding with medicated feed).

[0011] A2: The dosage is at least 0.1 μg / μL (suitable for administration methods after solution preparation).

[0012] 3. Administration methods and corresponding dosage forms:

[0013] (1) When feeding with medicated feed, the dosage forms of the drug can include tablets, powders, granules, capsules or sustained-release agents, and the application dosage of heparan sulfate is at least 10 mg / kg.

[0014] (2) After solution preparation, one of the following administration methods can be adopted:

[0015] B1: Injection administration.

[0016] B2: Drug immersion administration.

[0017] B3: Feeding with medicated feed (although the administration method is the same as that of A1 at this time, the dosage and dosage form may vary due to solution preparation, so it is listed separately to clarify the application scenario).

[0018] In the second aspect, the present invention provides the application of heparan sulfate in the preparation of an additive for preventing and treating grass carp reovirus.

[0019] In some specific embodiments, the application includes:

[0020] 1. The dosage of heparan sulfate as an additive in the feed is at least 10 mg / kg.

[0021] 2. When the additive is in solution form, the dosage of heparan sulfate in the additive is at least 0.1 μg / μL.

[0022] The beneficial effects of the present invention at least include the following:

[0023] (1) The present invention first proves at the cellular level that heparan sulfate exhibits excellent antiviral performance at extremely low concentrations (≥0.1 μg / mL). It not only effectively inhibits the adsorption of grass carp reovirus (GCRV), but also significantly hinders the subsequent infection process of the virus. This discovery provides a new perspective and strategy for the prevention and treatment of viral diseases in aquaculture.

[0024] (2) Through the artificial infection challenge experiment, the present invention further proves the antiviral effect of heparan sulfate in the actual aquaculture environment. After feeding grass carp infected with grass carp reovirus with feed containing extremely low concentrations of heparan sulfate (≥10 mg / kg), not only the survival rate of the grass carp population is significantly improved, but also the virus copy number in the fish tissue is significantly reduced, and the tissue damage caused by virus infection is improved.

[0025] (3) As an antiviral feed additive, heparan sulfate in the present invention has the advantages of low cost, low dosage, high safety, and broad application prospects. Its application in actual aquaculture is expected to bring significant economic and social benefits to the aquaculture industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0027] Figure 1 It is a diagram of the immunofluorescence results of the virus after treatment with heparan sulfate (HS) in the embodiments of the present invention; among them, the red particles represent the virus particles adsorbed on the cell surface, and the blue fluorescence is DAPI staining, which is used to label the cell nucleus; Control represents the control group; Heparan sulfate represents the heparan sulfate treatment group; Merge is the merged diagram, which merges the red fluorescence of GCRV and the blue fluorescence of DAPI for display, more intuitively presenting the relationship between the virus particles and the cells;

[0028] Figure 2 It is a diagram of the fluorescence quantitative PCR results after treatment with heparan sulfate in the embodiments of the present invention; among them, the horizontal axis represents the concentration of heparan sulfate (μg / mL); ** in the figure indicates extremely significant differences; compared with each other, after treatment with different concentrations of heparan sulfate, the decrease in the expression levels of VP5 and VP7 genes has extremely significant statistical significance;

[0029] Figure 3 It is a diagram of the Western blotting results after treatment with heparan sulfate in the embodiments of the present invention;

[0030] Figure 4 It is a survival rate curve diagram of feeding feed containing heparan sulfate (HS) or ordinary feed after infection with GCRV in the embodiments of the present invention; among them, "Con" represents the control group, injecting PBS and feeding ordinary grass carp feed; "GCRV" represents the group only injecting GCRV virus solution and feeding ordinary grass carp feed; "HS-10mg", "HS-100mg", and "HS-200mg" respectively represent the groups feeding feed containing 10mg / kg, 100mg / kg, and 200mg / kg heparan sulfate after injecting GCRV virus solution;

[0031] Figure 5In the embodiments of the present invention, it is a bleeding symptom diagram of grass carp infected with GCRV and fed with heparan sulfate-containing feed or ordinary feed;

[0032] Figure 6 In the embodiments of the present invention, it is a quantitative PCR result diagram of the relative copy number of the virus in grass carp infected with GCRV and fed with heparan sulfate-containing feed or ordinary feed;

[0033] Figure 7 In the embodiments of the present invention, it is a histopathological observation diagram of grass carp infected with GCRV and fed with heparan sulfate-containing feed or ordinary feed;

[0034] Figure 8 In the embodiments of the present invention, it is a quantitative PCR result diagram of 4 immune genes in grass carp infected with GCRV and fed with heparan sulfate-containing feed or ordinary feed. Detailed implementation manners

[0035] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below. Obviously, the described embodiments are some but not all of the embodiments of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present invention. For those conditions not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. For those reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase. The acquisition routes of various biological materials described in the embodiments only provide an experimental acquisition route to achieve the specific disclosure purpose, and should not be a limitation on the source of biological materials of the present invention. In fact, the sources of the biological materials used are extensive, and any biological materials that can be obtained without violating laws and moral ethics can be replaced and used according to the prompts in the embodiments.

[0036] In some specific embodiments of the present invention, the materials, reagents and instruments involved include but are not limited to the following:

[0037] Table 1 Materials, reagents and instruments

[0038]

[0039] In some specific embodiments of the present application, the meanings of the terms include but are not limited to the following:

[0040] 1. Grass carp kidney cells (CIK) are not only a sensitive model for GCRV replication in the drug experiment of grass carp reovirus (GCRV), used to observe the replication process of the virus in cells, but also an important tool for evaluating the antiviral effect of drugs. By comparing indicators such as virus titers between the drug-treated group and the untreated group, drugs with antiviral activity can be effectively screened and identified.

[0041] 2. Multiplicity of Infection (MOI) refers to the ratio of the number of viruses that can infect cells to the total number of cells. MOI describes the proportional relationship between the number of viruses that can infect cells and the total number of cells in a specific system, and can also be understood as the average number of cells infected by viruses per cell.

[0042] Example 1 In vitro inhibition of GCRV virus adsorption and infection by heparan sulfate

[0043] 1. Method

[0044] (1) Cell culture and virus infection

[0045] Dissolve heparan sulfate in PBS to prepare a stock solution with a concentration of 100 mg / μL for later use.

[0046] After trypsin digestion, grass carp kidney cells (CIK) were passaged into six-well plates and cultured using M199 medium containing 10% FBS (v / v). The six-well plates were placed in an incubator at 28 °C and 5% CO 2 and statically cultured for 24 h. Different concentrations of heparan sulfate solutions (final concentrations of 0, 0.1, 1, 10, 100 μg / μL) were added to the cells in the six-well plates and incubated at 28 °C for 1 h. Among them, the group with a concentration of 0 served as the control group without adding heparan sulfate.

[0047] After incubation, 100 MOI units of GCRV virus solution (Type I GCRV strain) were added to each group of cells and infected at 4 °C for 1 h.

[0048] (2) Immunofluorescence detection of virus particles on the cell surface

[0049] Collect the cells after incubation at 4 °C for 1 h, and incubate them at room temperature for 2 h using antibodies against the outer capsid proteins VP5 and VP7 of Type I GCRV (Grass Carp Reovirus, Strain Type I) (dilution volume ratio of 1:200) (v / v).

[0050] Rinse with PBS three times, 5 min each time. Incubate at room temperature for 1 h using Alexa 594-conjugated goat anti-rabbit IgG antibody and FITC-conjugated heparan sulfate antibody (dilution volume ratio of 1:2000) (v / v). Rinse with PBS again three times, 5 min each time.

[0051] Stain the cell nuclei with DAPI for 15 min. After staining, observe using a confocal microscope.

[0052] (3) RNA extraction and detection of viral gene expression levels

[0053] Collect the cells incubated at 4°C for 1 h as described above, and extract total RNA using TRIzol reagent. Reverse transcription is performed using the HiScript III 1st Strand cDNA Synthesis Kit according to the kit instructions to prepare the reaction system and obtain the cDNA template. The reverse transcription program is as follows: incubate at 25°C for 5 min, 37°C for 45 min, 85°C for 5 s, and finally cool on ice.

[0054] Detect the expression levels of the I-type GCRV VP5 and VP7 coding genes by fluorescence quantitative PCR reaction to reflect the relative number of viruses adsorbed to the cell surface. The VP5 and VP7 quantitative primers are designed based on conserved sequences (NCBI accession numbers: AF239175.1 and HQ018819.1, respectively). The reaction system is prepared using Taq Pro Universal SYBR qPCR Master Mix (a dedicated premix for real-time fluorescence quantitative PCR), specifically: 10 μL SYBR qPCR Master Mix, 7.4 μL ddH2O, 1 μL cDNA template, 0.8 μL forward primer, and 0.8 μL reverse primer. The PCR reaction is carried out in a real-time fluorescence quantitative PCR instrument. The reaction program is as follows: pre-denature at 95°C for 2 min; denature at 95°C for 15 s, anneal at 56°C for 30 s, extend at 72°C for 30 s, for 40 cycles; collect fluorescence signals at 65 - 95°C. Each sample is set with 3 replicates, and the β-actin gene is used as an internal reference for normalization. The primer sequence information is shown in Table 2, and the relative expression levels of the genes are calculated using the 2 -△△Ct -ΔΔCt method.

[0055] (4) Detection of viral protein expression by Western blotting

[0056] Collect the cells infected at 28°C for 24 h, and lyse the cells on ice for 30 min using RIPA lysis buffer. After centrifugation to obtain the supernatant, SDS-PAGE gel electrophoresis is performed. The electrophoresis conditions are: 120 V for 30 min; 90 V for 60 min. Transfer the electrophoresed protein sample to a PVDF membrane and block it with 5% (m / v) skim milk at room temperature for 2 h.

[0057] Incubate overnight at 4°C using VP5 and VP7 antibodies (dilution volume ratio 1:1000) (v / v) and the internal reference actin antibody (dilution volume ratio 1:2000) (v / v) respectively. Rinse with PBST buffer for a total of 3 times, 5 minutes each time. Incubate at room temperature for 2 hours using the HRP-labeled antibody as the secondary antibody (dilution volume ratio 1:2000) (v / v). Rinse again with PBST for a total of 3 times, 5 minutes each time. Add ECL chemiluminescent solution and process for 1 minute, expose the film in a darkroom, and scan and photograph.

[0058] 2. Results

[0059] Immunofluorescence results: The results showed that after adding heparan sulfate to the cells in the six-well plate and incubating for 1 hour before conducting the GCRV infection experiment, the number of virus particles bound to the cell surface decreased significantly (see Figure 1 ). This indicates that heparan sulfate may interfere with the binding process between the virus and the cell surface. If the virus cannot effectively bind to the cell surface, it is difficult to enter the cell and carry out subsequent infection and replication, thus initially showing its antiviral potential.

[0060] Fluorescence quantitative PCR results: The results showed that the expression levels of the viral genes VP5 and VP7 decreased significantly after treatment with heparan sulfate and showed a concentration-dependent effect. Among them, when the concentration of heparan sulfate used was 0.1 μg / μL, there was a significant inhibitory effect (see Figure 2 ). VP5 and VP7 are important coding genes of type I GCRV. The decrease in their expression levels means that the replication and transcription processes of the virus are inhibited, further indicating that heparan sulfate has an inhibitory effect on the infection of GCRV.

[0061] Western blotting results: The results showed that the expression levels of the GCRV-encoded proteins VP5 and VP7 decreased significantly after treatment with heparan sulfate and also showed a concentration-dependent effect. Among them, when the concentration of heparan sulfate used was 0.1 μg / μL, there was a significant inhibitory effect (see Figure 3 ). Protein is the product of gene expression. The decrease in protein expression levels corresponds to the decrease in gene expression levels, further confirming from the protein level that heparan sulfate can inhibit the infection and replication of GCRV.

[0062] 3. Conclusions

[0063] The above results indicate that heparan sulfate can inhibit the adsorption and subsequent infection process of GCRV virus under extremely low concentration conditions, providing a new idea and method for the prevention and treatment of GCRV virus.

[0064] Table 2 Sequence information

[0065]

[0066]

[0067] Example 2 Application of Heparan Sulfate in Feed Additive against GCRV Virus

[0068] 1. Method

[0069] (1) Preparation of experimental fish and virus infection

[0070] Select about 750 grass carps with a body weight of about 5 g, divide them into 5 groups equally, and raise them in plastic barrels with a diameter of about 1 m. Keep the water temperature at about 28 °C, the dissolved oxygen content above 5 mg / mL, and the water body pH value controlled at about 7.5. Inject 100 μL of type II GCRV virus solution (Grass Carp Reovirus, Strain Type II) into the grass carps in 4 of the groups. The RNA copy number in the virus suspension is 2.97×10 3 copies / μL, and the remaining 1 group is used as the control group, which is injected with an equal dose of PBS.

[0071] Three days after infection, feed the 4 groups injected with the virus with ordinary grass carp feed and grass carp feed containing different doses of heparan sulfate (10 mg / kg, 100 mg / kg, 200 mg / kg) respectively. The control group not infected with the virus continues to be fed with ordinary grass carp feed. Feed continuously for 3 days, once in the morning and once in the evening every day, and the weight of the feed fed each time is about 2% of the fish body weight.

[0072] During the experiment, carefully record the number of dead individuals of grass carps in each group every day, and draw the survival curve of each group after the experiment. In addition, randomly select 5 fish from each group on the fifth day of infection, and take their liver and intestinal tissues for subsequent RNA extraction, virus copy number detection, and histopathological section observation.

[0073] (2) RNA extraction and virus copy number detection

[0074] Total RNA was extracted from liver and intestinal tissues using TRIzol reagent, and cDNA was obtained by reverse transcription using the HiScript III 1st Strand cDNA Synthesis Kit. The expression level of the type II GCRV VP4-encoding gene was detected by fluorescence quantitative PCR reaction to reflect the relative copy number of the virus in each group. The quantitative primer sequences of the VP4 gene were designed according to its conserved sequence (NCBI accession number: KF712480.1). Fluorescence quantitative PCR reaction was performed using Taq Pro Universal SYBR qPCR Master Mix. The reaction system and procedure were the same as in Example 1 and were carried out in a real-time fluorescence quantitative PCR instrument. Each sample was set with 3 replicates, and the β-actin gene was used as an internal reference for normalization. The sequence information of the amplification primers is shown in Table 2 in detail, and the relative expression level of the gene was calculated using the 2 -△△Ct -method.

[0075] (3) Observation of tissue pathological sections

[0076] The liver and intestinal tissues of the above 5 groups were fixed with Bouin's fixative. After dehydration, clearing, wax infiltration, embedding and other steps, the embedded paraffin blocks were cut into thin sections and attached to glass slides. After dewaxing and hydration of the sections, they were successively placed in hematoxylin and eosin staining solutions for staining. Finally, the sections were observed under a microscope and photographed.

[0077] (4) Quantitative detection of immune-related genes

[0078] The expression levels of four immune-related genes, interferon regulatory factor 3 (IRF3), interferon regulatory factor 7 (IRF7), interferon 1 (IFN1), and interferon 3 (IFN3), in the liver tissues of each group were detected by fluorescence quantitative PCR reaction to reflect the immune level of the body in each group after virus infection. The quantitative primer sequences of the above four genes were designed according to their conserved sequences (NCBI accession numbers: KC898261.1, GQ141741.1, DQ357216.1, KU182642.1). The reaction system and procedure were the same as in Example 1 and were carried out in a real-time fluorescence quantitative PCR instrument. Each sample was set with 3 replicates, and the β-actin gene was used as an internal reference for normalization. The sequence information of the amplification primers is shown in Table 2 in detail, and the relative expression level of the gene was calculated using the 2 -△△Ct -method.

[0079] 2. Results

[0080] (1) Survival curve and mortality analysis

[0081] As Figure 4As shown, the mortality rate of the group injected with GCRV virus but fed with ordinary grass carp feed was as high as 86.67%. The mortality rates of the three groups injected with GCRV but fed with heparin sulfate-containing feed were 57.86% (10 mg / kg), 43.57% (100 mg / kg), and 52.86% (200 mg / kg), respectively. In addition, no dead individuals were found in the control group injected with PBS.

[0082] The in vivo experiment of grass carp infected with GCRV directly shows that heparin sulfate can significantly reduce the mortality rate of grass carp after infection with GCRV, strongly proving its antiviral effect in vivo and improving the survival ability of grass carp.

[0083] (2) Detection of virus copy number

[0084] The results of fluorescence quantitative PCR showed that the virus copy number was significantly reduced in the groups fed with heparin sulfate-containing feed. Among them, heparin sulfate in the feed could significantly inhibit the replication of GCRV at a very low concentration condition (10 mg / kg) Figure 6 ). This further confirmed from the perspective of virus load that heparin sulfate can inhibit the proliferation of GCRV in vivo, reduce the number of viruses in the host, and thus reduce the degree of virus infection.

[0085] (3) Observation of tissue pathological sections

[0086] The results of observation of tissue pathological sections showed that severe necrotic lesions, cell vacuolization, and nuclear rupture were observed in the liver and intestinal tissues of the group injected with GCRV but fed with ordinary grass carp feed. The pathological changes in the liver and intestinal tissues of the group fed with heparin sulfate-containing feed were significantly reduced. Among them, heparin sulfate in the feed could significantly improve the tissue pathological effect after GCRV infection at a very low concentration condition (10 mg / kg) Figure 7 ). It can be seen that heparin sulfate can reduce the damage of virus infection to grass carp tissues, protect the normal structure and function of tissues and organs, and further prove its antiviral effectiveness.

[0087] (4) Expression levels of immune-related genes

[0088] The results of fluorescence quantitative PCR showed that the expression levels of 4 immune genes were significantly reduced in the groups fed with heparin sulfate-containing feed. Among them, heparin sulfate in the feed could significantly inhibit the expression levels of the 4 genes at a very low concentration condition (10 mg / kg) Figure 8 ). It can be seen that heparin sulfate can reduce the damage caused by the excessive immune inflammatory response after virus infection to the body, and thus promote the survival of the body.

[0089] 3. Conclusion

[0090] In summary, heparan sulfate can effectively function at extremely low concentrations as an antiviral feed additive, significantly reducing the mortality rate of grass carp after GCRV infection and alleviating the damage to grass carp tissues caused by virus infection. Considering the comprehensive results of in vitro and in vivo experiments, from virus binding and replication inhibition at the cellular level to mortality reduction, virus load decrease, and tissue damage alleviation at the whole animal level, a relatively complete evidence chain is formed, demonstrating that heparan sulfate can be used as an antiviral feed additive against GCRV. This provides strong experimental evidence and potential market value for the application of heparan sulfate in fishery aquaculture.

[0091] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. Application of heparan sulfate in the preparation of drugs for preventing and treating grass carp reovirus.

2. The application according to claim 1, characterized in that: Heparan sulfate as an active ingredient in drugs for the prevention and treatment of grass carp reovirus.

3. The use according to claim 1 or 2, characterized in that: The drug also includes pharmaceutically acceptable excipients.

4. The use according to claim 1 or 2, characterized in that: The dosage of heparan sulfate in the medicine includes one of the following: A1. The dose is at least 10 mg / kg; A2. The dose is at least 0.1 μg / μL.

5. The use according to claim 4, characterized in that: The drug is administered by mixing with feed, and the application dosage of heparan sulfate is at least 10 mg / kg.

6. The use according to claim 5, characterized in that: The dosage form of the drug includes tablets, powders, granules, capsules or sustained-release preparations.

7. The use according to claim 4, characterized in that: The heparan sulfate is prepared into a solution, wherein the dosage of the heparan sulfate is at least 0.1 μg / μL; and then administered in one of the following ways: B1. Injection; B2. Drug immersion administration; B3. Mix feed and feed.

8. Application of heparan sulfate in the preparation of additives for preventing and controlling grass carp reovirus.

9. The use according to claim 8, characterized in that: The dosage of heparan sulfate as an additive in feed is at least 10 mg / kg.

10. The use according to claim 8, characterized in that: The additive is a solution-type additive, and the dosage of heparan sulfate in the additive is at least 0.1 μg / μL.

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