Application of fumaric acid in preventing and treating grass carp reovirus infection

Abstract

The present invention relates to the field of aquaculture. The present invention discloses a new use of fumaric acid in the preparation of a drug for preventing and treating grass carp reovirus (GCRV) infection. The present invention studies show that fumaric acid exhibits significant antiviral efficacy against GCRV in both in vitro and in vivo experiments. Fumaric acid not only effectively inhibits the replication of GCRV in a cell culture system, significantly reduces the viral load, but also promotes the expression of immune-related factors and enhances the immune defense mechanism of host cells. In addition, the present invention confirms through an artificial infection and toxicity challenge experiment that the average survival rate of the grass carp group in the fumaric acid treatment group can be significantly increased by 19.60 percentage points compared with the untreated group when facing GCRV infection. In an experiment simulating a natural breeding state, regular feeding of feed containing fumaric acid successfully prevented the outbreak of grass carp hemorrhagic disease. The present invention explores the application potential of fumaric acid in the fields of antiviral and immune enhancement for the first time, and opens up a new way for the multifunctional application of fumaric acid in the field of aquaculture.

Description

Technical Field

[0001] The present invention relates to the field of aquaculture, and in particular to application of fumaric acid in preventing and treating grass carp reovirus infection. Background Art

[0002] Currently, grass carp aquaculture in my country accounts for approximately 20% of the total aquaculture output, and annual production losses due to diseases account for over 30% of the total annual grass carp production. Among grass carp pathogens, grass carp reovirus (GCRV) poses the greatest threat. Grass carp hemorrhagic disease, caused by infection with GCRV, is highly contagious and lethal, leading to massive production losses and long considered one of the most prominent problems in my country's freshwater aquaculture. Grass carp reovirus particles exhibit icosahedral symmetry, possess a double-layered capsid, lack an envelope, and have a genome composed of 11 double-stranded RNA strands. Grass carp reovirus infection can cause varying degrees of congestion and hemorrhage in various organs and tissues of grass carp, with a mortality rate exceeding 80%. Currently, there is no ideal treatment for grass carp reovirus infection, and prevention is the primary focus. Grass carp hemorrhagic disease, caused by GCRV, is a major viral disease threatening the grass carp aquaculture industry. Grass carp reovirus can be cultured in vitro in a variety of grass carp cell lines, such as grass carp kidney cells (CIK), grass carp fin ray cells (CF), grass carp ovary cells (GCO) and grass carp blastocyst cells (GCB), and produce significant cytopathic effects.

[0003] At present, the prevention and control of GCRV mainly focuses on vaccine research. With the development of genetically engineered vaccines, various grass carp hemorrhagic disease vaccines, represented by subunit vaccines and nucleic acid vaccines, have emerged one after another. Currently, various vaccines are mainly used by injection, oral administration or immersion. However, the large-scale production and application of vaccines are still restricted, mainly because the strong specificity of vaccines makes it impossible to apply to viruses that are prone to mutation. Based on this, finding anti-GCRV drugs with good antiviral effects has significant advantages over vaccine prevention and control in terms of maintaining antiviral effects on different strains. However, there is currently no specific drug for grass carp hemorrhagic disease. Even the commonly used hemorrhagic disease prevention and control drugs in the early days, such as ribavirin, have carcinogenic side effects and are now banned. Therefore, it is crucial to screen for a drug with low side effects to prevent and treat GCRV infection. Summary of the Invention

[0004] In view of this, the present invention carried out DIA proteomics and non-targeted metabolomics analysis on five tissues, namely gills, liver, intestines, kidneys and muscles before and after GCRV infection, and found that carbohydrate metabolism is the metabolic pathway that is mainly affected after GCRV infection. Then, the butyrate metabolic pathway belonging to carbohydrate metabolism in kidney tissue was further studied, and it was found that the metabolic intermediate fumaric acid therein has an antiviral effect. Based on this, the present invention studied the feasibility of fumaric acid in preventing and treating GCRV infection, and found that fumaric acid has the effect of significantly inhibiting GCRV replication and can induce cellular immune enhancement. In addition, the present invention also found that fumaric acid can significantly improve the survival rate of grass carp juveniles after GCRV infection.

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

[0006] In a first aspect, the present invention provides use of fumaric acid in preparing a medicament for preventing and treating grass carp reovirus infection.

[0007] In a second aspect, the present invention provides the use of fumaric acid in preparing a preparation for preventing and treating grass carp reovirus infection.

[0008] In a third aspect, the present invention provides use of fumaric acid in preparing a preparation for inhibiting the replication of grass carp reovirus in cells.

[0009] In some preferred embodiments, the preparation comprises fish feed, which is a dry granular feed or a fermented liquid feed.

[0010] In some preferred embodiments, the amount of fumaric acid added to the feed is 1 wt % of the feed.

[0011] In a fourth aspect, the present invention provides use of fumaric acid in the preparation of a medicament for inhibiting the replication of grass carp reovirus in cells.

[0012] In a fifth aspect, the present invention provides the use of fumaric acid in the preparation of an immunopotentiator for grass carp reovirus-infected cells.

[0013] In some preferred embodiments, the amount of fumaric acid added to the culture medium of the cells is 0.6-0.8 mg / mL.

[0014] Furthermore, the grass carp reovirus includes at least one of type I strain and type II strain.

[0015] The beneficial effects of the present invention include at least the following:

[0016] The present invention uses data-independent acquisition (DIA) proteomics and non-targeted metabolomics to perform two types of sequencing on virus-infected tissue samples. Combined with bioinformatics analysis and biostatistical analysis of the data, metabolite molecules with extremely significant downregulated expression are identified, thereby screening for candidate GCRV prevention and treatment preparations or drugs, namely fumaric acid.

[0017] This study reveals for the first time that fumaric acid has significant antiviral effects against GCRV at the cellular and individual levels. Specifically, fumaric acid exhibited excellent antiviral activity in cell culture systems within a concentration range of 0.6-0.8 mg / mL. It not only effectively inhibited GCRV replication and significantly reduced viral load, but also promoted the expression of immune-related factors and enhanced the host cell's immune defense mechanism. This provides a new perspective and approach to viral disease prevention and control strategies in the aquaculture industry.

[0018] The present invention also confirmed the antiviral effect of fumaric acid in an actual breeding environment through artificial infection and virus attack experiments. That is, when the grass carp group in the fumaric acid-treated group faced GCRV infection, its average survival rate was significantly increased by 19.60 percentage points compared with the untreated group. This result directly and strongly proves the effectiveness of fumaric acid in improving the antiviral ability and breeding survival rate of fish.

[0019] In addition, in experiments simulating natural breeding conditions, regular feeding of feed containing fumaric acid successfully prevented the outbreak of grass carp hemorrhagic disease, further confirming its stability and practicality in complex ecological environments. As a widely recognized feed additive, fumaric acid has a large amount of literature supporting its safety and functionality in promoting fish growth. This invention reveals for the first time the potential value of fumaric acid in the antiviral field, and its recommended usage (1wt%) is within the known safe range (0.5-2wt%) and will not have a negative impact on the aquatic environment, opening up new avenues for the multifunctional application of fumaric acid in aquaculture.

[0020] Therefore, given fumaric acid's significant role in inhibiting viral infection and enhancing immunity, this invention is expected to provide a new method for preventing and treating viral and other diseases in aquaculture. As a naturally occurring organic acid, fumaric acid also meets the requirements of green aquaculture technology. Furthermore, given fumaric acid's dual effects of promoting growth and combating viruses, it may be possible to develop it into a multifunctional feed additive in the future, providing a more comprehensive and efficient solution for the aquaculture industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0022] Figure 1 The results of GCRV 873S6 gene expression detection under different concentrations of fumaric acid treatment in the embodiment of the present invention are shown;

[0023] Figure 2 The results of the detection of the gene expression of immune-related factors (Figure A is the interferon family members IFN1 and IFN3, Figure B is the interferon regulatory factors IRF3 and IRF7) treated with different concentrations of fumaric acid in the examples of the present invention;

[0024] Figure 3 The survival rate statistics of the fumaric acid group, the positive drug group (astragalus polysaccharide group, five yellow powder group) and the control group (blank control) in the GCRV artificial infection challenge experiment in the embodiment of the present invention are as follows;

[0025] Figure 4 These are the histopathological test results of the liver, spleen, and kidney in the GCRV artificial infection challenge experiment in the embodiment of the present invention. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical solutions and advantages of this application more clearly understood, the present application is further described in detail below with reference to the following examples. It should be understood that the specific examples described herein are merely for the purpose of explaining this application and are not intended to limit this application. Reagents not described in detail in this application are all conventional reagents and can be obtained from commercial channels; methods not specifically described in detail are all conventional experimental methods and can be obtained from the prior art.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which this invention pertains. The terms used in this specification are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The terms "including," "having," and any variations thereof, are intended to cover non-exclusive inclusions.

[0028] Example 1 Drug Screening for Preventing and Treating GCRV Infection

[0029] 1. Proteome and metabolome sequencing

[0030] 1) Experimental grouping and sample tissue collection and processing

[0031] A six-month-old grass carp full-sib group (15±3cm, 40±10g, 100 fish) was used as the experimental group and was challenged with GCRVHZ08 by gavage. The viral copy number was 7.42×10 8 The oral dose was 500 μL / tail. After infection, samples were collected when the grass carp showed obvious symptoms such as solitary swimming, unbalanced swimming and surface bleeding. Five tissues, including gills, liver, intestines, kidneys and muscles, were collected from each fish. 100 healthy grass carp with the same genetic background served as the control group, and the above tissue samples were collected at the same time points. The same tissue samples of every three fish in the experimental group and the control group were mixed into one sample to be sequenced. Three parallel samples were set up for each sample to be sequenced, and a total of 30 samples (5×3×2) were obtained from the experimental group and the control group.

[0032] 2) Proteome sequencing and analysis

[0033] DIA proteomics analysis of 30 samples was performed using a Shimadzu LC-20AD liquid phase system (Shimadzu, Japan). DDA library construction and DIA sample analysis were performed on an UltiMate 3000 UHPLC (Thermo Fisher Scientific, San Jose, CA) and an Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific, San Jose, CA). MaxQuant software (version 1.5.3.30) was used to construct a library for DIA data and used as the spectral library for DIA analysis. Spectronaut software was used to analyze DIA data, and iRT peptides were used to correct retention times. False positive control was then performed using a target-decoy model suitable for SWATH-MS with an FDR of 1%. Error correction and normalization were performed for each sample, and significantly differentially expressed proteins were screened based on a fold change (FC) ≥ 2 and a P < 0.05.

[0034] The results showed that a total of 114,015 peptides and 10,808 proteins were quantified from 30 samples. The up-regulated proteins in gills, liver, kidney and muscle tissues were significantly more than the down-regulated proteins, while the down-regulated proteins in the intestine were more than the up-regulated proteins. The kidney had the most differentially expressed proteins, with 1,529 up-regulated proteins and 219 down-regulated proteins.

[0035] 3) Metabolome sequencing and analysis

[0036] Untargeted metabolomic UPLC-MS analysis of 30 samples was performed using a Waters 2777C UPLC (Waters, USA) coupled to a Q Exactive HF high-resolution mass spectrometer (Thermo Fisher Scientific, USA) in ESI mode for metabolite separation and detection. Off-line mass spectrometry data were imported into CompoundDiscoverer 3.3 (Thermo Fisher Scientific, USA) and analyzed using the BMDB, mzCloud, and ChemSpider databases. A data matrix containing metabolite peak areas and identification information was generated. The results exported from Compound Discoverer were then imported into metaX for data preprocessing. Probabilistic Quotient Normalization (PQN) was first used to normalize the data to obtain relative peak areas. Batch effects were corrected using QC-RLSC (Quality Control-based Robust LOESS signal correction). Finally, compounds with a relative peak area coefficient of variation (CV) greater than 30% in all QC samples were removed. Fold change and t-test were used to calculate P values ​​using univariate analysis. Significantly differential metabolites were screened based on FC ≥ 1.2 or ≤ 0.83 and P < 0.05.

[0037] The results showed that a total of 4040 metabolites were identified, and the kidney also had the most differentially expressed metabolites, with 646 up-regulated and 875 down-regulated metabolites, respectively.

[0038] 2. Screening of metabolic molecules in key pathways

[0039] 1) KEGG enrichment analysis and common pathway identification

[0040] KEGG enrichment analysis was performed on the DEPs and DEMs of each tissue at the protein and metabolite levels, and the pathways with P < 0.05 were selected and the intersection was taken to obtain the common pathways of each tissue at the protein and metabolic levels.

[0041] A total of 16 pathways were identified, including carbohydrate metabolism (6) and some immune-related amino acid metabolism (4). Other pathways included lipid metabolism (2), prosthetic group and vitamin metabolism (2), energy metabolism (1), and transporter catabolism (1). The results showed that GCRV infection mainly affected changes in host carbohydrate metabolism pathways.

[0042] 2) In-depth analysis of differential metabolic pathways in the kidney

[0043] In-depth analysis of the kidneys with the most differentially expressed proteins and metabolites revealed that only butyrate metabolism, a metabolic pathway within carbohydrate metabolism, was enriched in 14 DEPs and DEMs within the butyrate metabolism pathway, seven of which were concentrated in the tricarboxylic acid (TCA) cycle. For example, upregulation of 4-aminobutyrate aminotransferase (FC=2.43, P<0.05) and glutamate decarboxylase 1 (FC=4.87, P<0.0001), which contribute to the TCA cycle, was observed. TCA cycle intermediates α-ketoglutarate (FC=7.04, P<0.001) and succinate (FC=1.48, P<0.05) were also upregulated, while downregulation of fumarate (FC=0.55, P<0.0001), maleate (FC=0.54, P<0.01), and pyruvate (FC=0.47, P<0.05) was observed.

[0044] 3. Screening results: The proteomic and metabolomic data of the present invention showed that fumarate in the renal butyrate metabolism was downregulated during GCRV infection (P = 7.33 × 10 -5 ), compared with other metabolites such as maleic acid (P = 1.09 × 10 -3 ) and pyruvate (P = 1.41 × 10 -2 ), the P value of fumaric acid was significantly lower and more reliable, so fumaric acid was selected as a candidate drug for the prevention and treatment of GCRV infection.

[0045] Example 2 Application of fumaric acid in antiviral and immune enhancement functions in cells

[0046] 1) Cell culture, fumaric acid treatment, and viral challenge

[0047] Fumaric acid (Cat. No. S30198-500g) was purchased from Shanghai Yuanye Biotechnology Co., Ltd. CIK cells (grass carp kidney cells) were seeded in 6-well plates. When the growth area reached approximately 80% on the second day, the cells were cultured with complete medium containing different final concentrations of fumaric acid (0, 0.1, 0.2, 0.4, 0.6, and 0.8 mg / mL) for 4 h. DMSO with 0 mg / mL dissolved fumaric acid was used as the control group. After 4 h, 100 μL of GCR V 873 virus solution (3.76×10 7 The cells were challenged with 500 copies / mL of 500 μg / mL and the cells were collected 24 h after the challenge.

[0048] 2) RNA extraction and gene expression detection

[0049] The collected cell samples were all extracted with Trizol (Fisher Scientific, USA) and the reverse transcription reaction was completed using the HiScript II 1st Strand cDNA Synthesis Kit (Vazyme, China). The expression levels of the GCRV 873S6 gene and interferon system-related genes (IFN1, IFN3, IRF3 and IRF7) were then detected using the qPCR method. The reaction system was configured using HiScript IIQ RT SuperMix for qPCR+gDNA wiper (Vazyme, China). The PCR reaction was performed in a real-time fluorescence quantitative PCR instrument (CF X96, Bio Rad). Each sample was set up with 3 replicates, and the β-actin gene was used as an internal reference to normalize gene expression. The primer sequences for amplifying these genes are shown in Table 1. The relative expression levels of the genes were calculated using 2 -△△Ct Method calculation.

[0050] Table 1 qPCR primer sequences

[0051]

[0052]

[0053] 3) Analysis of GCRV infection outcomes and immune-related factor gene expression

[0054] like Figure 1 As shown, the qPCR results showed that 24 hours after GCRV 873 infection, the content of GCRV 873 in the high concentration fumaric acid treatment group (0.6 mg / mL, 0.8 mg / mL) was significantly decreased compared with the control group (P<0.0001), while the content of GCRV 873 in the low concentration fumaric acid treatment group (0.1 mg / mL, 0.2 mg / mL, 0.4 mg / mL) did not change significantly, indicating that high concentration fumaric acid has anti-GCRV function in in vitro experiments.

[0055] The results are as follows Figure 2 As shown in the data, high concentrations of fumaric acid (0.6 mg / mL, 0.8 mg / mL) can simultaneously induce the upregulation of IFN1, IFN3, IRF3 and IRF7 gene expression, among which IFN1 and IRF7 expression levels are higher, while low concentrations of fumaric acid (0.1 mg / mL, 0.2 mg / mL) inhibit the expression of IFN and IRF, which is basically opposite to the gene expression changes of GCRV 873 in cells. This once again shows that the concentration of fumaric acid is the key to its inhibition of GCRV infection, and this effect may also be mediated by the interferon system.

[0056] 4) Conclusion

[0057] Fumaric acid demonstrated significant anti-GCRV 873 virus activity in in vitro experiments, and was significantly affected by its concentration. High concentrations of fumaric acid (0.6 mg / mL and 0.8 mg / mL) were able to significantly inhibit viral replication. In addition, fumaric acid may exert its antiviral effect through immune enhancement. High concentrations of fumaric acid can induce upregulation of the expression of genes such as IFN1, IFN3, IRF3, and IRF7, thereby enhancing the antiviral ability of cells. The changes in the expression of IFN1, IFN3, IRF3, and IRF7 genes showed a basically opposite trend to the changes in the GCRV873 virus content, further supporting the possibility that fumaric acid exerts its antiviral effect through immune enhancement.

[0058] Example 3 Application of Fumaric Acid in Antiviral Preparations

[0059] 1. Artificial infection and virus challenge experiment

[0060] 1) Experimental grouping, feed additive configuration and feeding management

[0061] In an indoor cement tank, 24 cages were configured to divide the water body into 24 compartments. Fifty grass carp (13±4 cm, 35±11 g) were placed in each cage and cultured in a continuous flow system with adequate dissolved oxygen. The 24 cages were assigned to three treatments: a normal diet, a fumaric acid group (0.5%, 1%, and 2% of the diet weight), an astragalus polysaccharide group (0.1% and 1% of the diet weight), and a five-yellow powder group (0.5% and 1% of the diet weight). Each treatment concentration was replicated in triplicate. Astragalus polysaccharide and five-yellow powder, two conventional feed enhancers, served as positive control groups. The diet used was 2-3 mm, 30% protein extruded pellets purchased from Tongwei Co., Ltd. Grass carp were fed the four treatments for 14 consecutive days, once in the morning and evening, with the daily feed intake representing approximately 2% of their body weight.

[0062] 2) Artificial infection, histopathological examination and survival rate statistics

[0063] After 14 days, each fish was gavaged with GCRVHZ08 virus solution (7.42×10 8 After the challenge, the mortality of fish in each group was recorded every day. The diseased fish in the normal feed group and the fumaric acid group (1%) were collected and the liver, spleen and kidney tissues were taken for pathological section observation. Figure 3As shown, the average survival rate of the normal feed group was 4.08%, the survival rates of the fumaric acid groups (0.5%, 1% and 2%) were 10.00%, 23.68% and 20.67%, respectively, the survival rates of the astragalus polysaccharide groups (0.1% and 1%) were 7.33% and 6.67%, and the average survival rates of the five-yellow powder groups (0.5% and 1%) were 9.33% and 8.67%. The survival rate of the fumaric acid group (1%) was 14.35-19.60 percentage points higher than that of the control group and the positive drug group.

[0064] Histopathological observations Figure 4 As shown in the results, after adding fumaric acid to the feed, the damage of grass carp tissues infected with GCRV was significantly reduced, and the degree of nuclear pyknosis and intercellular space in the liver, the intercellular space in the spleen, and the degree of tubular contraction and the number of monocytes in the kidney were all smaller than those in the normal feed group.

[0065] 3) Conclusion: Compared with the normal feed group, the average survival rate of grass carp in the fumaric acid group was significantly improved (from 4.08% to 23.68%), indicating that fumaric acid plays an important protective role in grass carp's resistance to GCRV infection and can significantly improve the survival ability of fish. Histopathological observations further confirmed the protective effect of fumaric acid. In the fumaric acid group, the damage to key organs such as the liver, spleen and kidneys caused by GCRV infection was significantly reduced, indicating that fumaric acid can alleviate the damage of the virus to fish tissues. Compared with the astragalus polysaccharide and five-yellow powder groups, the fumaric acid group also had a higher survival rate, showing its superiority in improving the antiviral ability of grass carp.

[0066] 2. Simulating natural breeding state experiment

[0067] In three outdoor ponds (1 mu, 1 mu, and 2.5 mu) where grass carp hemorrhagic disease broke out in 2022, one-month-old grass carp fry (2,000 per mu) were gradually stocked starting in June 2023. The water temperature was then monitored daily. When the water temperature exceeded 27°C, fumaric acid was added to the feed at 1% of the feed weight and fed to the fish for 14 consecutive days, morning and evening. By December 2023, no grass carp in the three ponds had experienced hemorrhagic disease. The results showed that adding fumaric acid to the feed can effectively prevent the outbreak of grass carp hemorrhagic disease.

[0068] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Use of fumaric acid in the preparation of drugs for preventing and treating grass carp reovirus infection.

2. Use of fumaric acid in the preparation of preparations for preventing and treating grass carp reovirus infection.

3. Use of fumaric acid in the preparation of a preparation for inhibiting the replication of grass carp reovirus in cells, characterized in that: In the culture medium of the cells, the added amount of fumaric acid is 0.6-0.8 mg / mL.

4. Use of fumaric acid in the preparation of a drug for inhibiting the replication of grass carp reovirus in cells, characterized in that: In the culture medium of the cells, the added amount of fumaric acid is 0.6-0.8 mg / mL.

5. Use of fumaric acid in the preparation of an immunopotentiator for grass carp reovirus-infected cells, characterized in that: In the culture medium of the cells, the added amount of fumaric acid is 0.6-0.8 mg / mL.

6. The use according to any one of claims 1 to 5, characterized in that The grass carp reovirus includes at least one of a type I strain and a type II strain.

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