Application of fumaric acid in prevention and treatment of grass carp reovirus infection

Abstract

The invention relates to the field of aquaculture. The invention discloses a novel application of fumaric acid in preparation of a medicine for preventing and treating grass carp reovirus (GCRV) infection. Researches show that fumaric acid shows remarkable antiviral efficacy on GCRV in both in-vitro experiments and in-vivo experiments. Fumaric acid in a cell culture system not only effectively inhibits the replication of GCRV and remarkably reduces the virus load, but also promotes the expression of immune related factors and enhances the immune defense mechanism of host cells. Besides, an artificial infection challenge experiment proves that the average survival rate of a grass carp group in a fumaric acid treatment group can be remarkably increased by 19.60% compared with an untreated group when the grass carp group is infected by GCRV (Growth Factor Receptor Virus). In an experiment for simulating a natural culture state, the hemorrhagic disease of the grass carp is successfully prevented from outbreak by regularly feeding the grass carp with a feed containing fumaric acid. According to the invention, the application potential of fumaric acid in the fields of virus resistance and immunological enhancement is explored for the first time, and a new way is opened up for multifunctional application of fumaric acid in the field of aquaculture.

Description

Technical Field

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

[0002] At present, the amount of grass carp cultured in my country accounts for about 20% of the total amount of cultured grass carp, and the annual production reduction caused by diseases accounts for more than 30% of the total annual grass carp cultured amount. Among the pathogens of grass carp, grass carp reovirus (GCRV) is the most harmful. The grass carp hemorrhagic disease caused by its infection is highly contagious and highly lethal, leading to large-scale production reduction. It has always been regarded as one of the most prominent problems in freshwater aquaculture in my country. Grass carp reovirus particles are icosahedral symmetric, have a double-layer capsid, no capsule, and the genome consists of 11 double-stranded RNAs. Grass carp reovirus infection can cause various organs and tissues of grass carp to have different degrees of congestion and bleeding, and the mortality rate is as high as more than 80%. At present, there is no ideal treatment for grass carp reovirus infection, and prevention is the main work. Grass carp hemorrhagic disease caused by grass carp reovirus is the main viral disease that endangers 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 treatment of GCRV mainly focuses on vaccine research. With the development of genetic engineering vaccine research, various grass carp hemorrhagic disease vaccines represented by subunit vaccines and nucleic acid vaccines have appeared one after another. At present, 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 easy to mutate. Based on this, finding anti-GCRV drugs with good antiviral effects has significant advantages over vaccine prevention and treatment in terms of maintaining antiviral effects on different strains. However, there is currently no specific drug for grass carp hemorrhagic disease, and 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 a drug with low side effects to prevent and treat GCRV infection. Summary of the invention

[0004] In view of this, the present invention has carried out DIA proteomics and non-targeted metabolomics analysis on five tissues of gill, liver, intestine, kidney and muscle 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 feasibility of fumaric acid in preventing and treating GCRV infection is studied, and it is found that fumaric acid has the effect of significantly inhibiting GCRV replication, and can induce cellular immune enhancement. In addition, the present invention also finds that fumaric acid can significantly improve the survival rate of grass carp fry after infection with GCRV.

[0005] The technical solution of the present invention is achieved in this way:

[0006] In a first aspect, the present invention provides the use of fumaric acid in the preparation of a drug 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 the 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 dry granular feed or 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 the use of fumaric acid in the preparation of a drug 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, in the culture medium of the cells, the added amount of fumaric acid is 0.6-0.8 mg / mL.

[0014] Furthermore, the grass carp reovirus includes at least one of a type I strain and a 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 omics sequencing on virus-infected tissue samples, combines bioinformatics analysis and biostatistics analysis of the data, identifies metabolite molecules with extremely significantly downregulated expression, and thus screens out candidate GCRV prevention and treatment preparations or drugs, namely fumaric acid.

[0017] This invention reveals for the first time that fumaric acid has a significant antiviral effect on GCRV at the cellular and individual levels. Specifically, fumaric acid exhibits excellent antiviral activity in a cell culture system within a concentration range of 0.6-0.8 mg / mL, which not only effectively inhibits the replication of GCRV and significantly reduces the viral load, but also promotes the expression of immune-related factors and enhances the immune defense mechanism of host cells, providing a new perspective and idea for the prevention and control strategy of viral diseases in aquaculture.

[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 treatment 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 powerfully proves the effectiveness of fumaric acid in improving the antiviral ability of fish and the survival rate of breeding.

[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 a complex ecological environment. As a widely recognized feed additive, fumaric acid has a lot of literature support for its safety and functionality in promoting fish growth. The present 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 safety range (0.5-2wt%), and will not have a negative impact on the water environment, opening up a new path for the multifunctional application of fumaric acid in aquaculture.

[0020] Therefore, in view of the significant role of fumaric acid in inhibiting viral infection and enhancing body immunity, the present invention is expected to provide a new method for preventing and treating viral diseases or other diseases in aquaculture. As a naturally occurring organic acid, fumaric acid also meets the requirements of green aquaculture technology. At the same time, combined with the dual role of fumaric acid in promoting growth and antiviral, it may be developed into a multifunctional feed additive in the future to provide 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 the description of 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 creative work.

[0022] Figure 1 The results of GCRV 873S6 gene expression detection under different concentrations of fumaric acid treatment in 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, and Figure B is the interferon regulatory factors IRF3 and IRF7) treated with different concentrations of fumaric acid in the embodiments 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 and toxicity challenge experiment in the embodiment of the present invention are shown;

[0025] Figure 4 These are the results of pathological examination of liver, spleen and kidney tissues in the GCRV artificial infection challenge experiment in the embodiments of the present invention. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application is further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described herein are only used to explain the present application and are not intended to limit the present application. The reagents not described separately in detail in the present application are all conventional reagents and can be obtained from commercial channels; the methods not 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 in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The terms "including" and "having" and any variations thereof of the present invention 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 challenged with GCRVHZ08 by oral gavage. The viral copy number was 7.42×10 8 copies / mL, and the oral dose was 500μL / tail. After the virus attack, samples were collected when the grass carp showed obvious symptoms such as solitary swimming, unbalanced swimming and surface bleeding after virus infection. Five tissues, including gills, liver, intestines, kidneys and muscles, were collected from each fish. 100 healthy grass carps with the same genetic background were used as the control group, and the above tissue samples were collected at the same time point. 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 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] The DIA proteome liquid phase separation of 30 samples was performed using the Shimadzu LC-20AD liquid phase system (Shimadzu, Japan). DDA library construction and DIA sample analysis were performed on the high performance liquid chromatograph UltiMate 3000UHPLC (Thermo Fisher Scientific, San Jose, CA) and mass spectrometer Orbitrap Exploris480 (Thermo Fisher Scientific, San Jose, CA). The DDA data were library-built using MaxQuant software (version 1.5.3.30) as the spectral library for DIA analysis. Spectronaut software was used to analyze DIA data, and iRT peptides were used to correct retention times. Then, based on the Target-decoy model applicable to SWATH-MS, false positive control was completed with FDR 1%. Error correction and normalization were performed for each sample, and significant differential proteins were screened based on Fold Change (FC) ≥ 2 and 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] The non-targeted metabolome UPLC-MS analysis of 30 samples was performed using a Waters 2777C UPLC (Waters, USA) in tandem with a Q Exactive HF high-resolution mass spectrometer (Thermo Fisher Scientific, USA) in ESI mode for the separation and detection of metabolites. The mass spectrometry data were imported into Compound Discoverer 3.3 (Thermo Fisher Scientific, USA) software, and after mass spectrometry data analysis combined with the BMDB database, mzCloud database, and ChemSpider online database, a data matrix containing information such as metabolite peak area and identification results was obtained. The results exported by Compound Discoverer were then imported into metaX for data preprocessing. First, the data were normalized using the Probabilistic Quotient Normalization (PQN) method to obtain the relative peak area; QC-RLSC (Quality control-based robust LOESS signal correction, local polynomial regression fitting signal correction) was used to correct the batch effect; finally, compounds with a relative peak area CV (Coefficient of Variation) greater than 30% in all QC samples were deleted. Fold change and t-test were used to calculate the P value using univariate analysis, and significant 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 DEP and DEM 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] The results showed that 16 pathways were obtained, including carbohydrate metabolism (6) and some immune-related amino acid metabolism (4), and others included lipid metabolism (2), prosthetic group and vitamin metabolism (2), energy metabolism (1) and transport catabolism (1). The results showed that GCRV infection mainly affected the changes in the host carbohydrate metabolism pathway.

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

[0043] The kidneys with the most differential proteins and metabolites were selected for in-depth analysis. Among the differential metabolic pathways in the kidney, only butyrate metabolism belongs to carbohydrate metabolism. There are 14 DEPs and DEMs in the enriched butyrate metabolic pathways, 7 of which are concentrated in the tricarboxylic acid cycle. For example, 4-aminobutyrate aminotransferase (FC=2.43, P<0.05) and glutamate decarboxylase 1 that promote TCA cycle were upregulated (FC=4.87, P<0.0001), α-ketoglutarate (FC=7.04, P<0.001) and succinate (FC=1.48, P<0.05) intermediates of TCA cycle were upregulated, and fumarate (FC=0.55, P<0.0001), maleate (FC=0.54, P<0.01) and pyruvate (FC=0.47, P<0.05) were downregulated.

[0044] 3. Screening results: The proteomic and metabolomic data of the present invention showed that fumaric acid in 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 virus challenge

[0047] Fumaric acid (Cat. No.: S30198-500g) was purchased from Shanghai Yuanye Biotechnology Co., Ltd. CIK cells (grass carp kidney cells) were inoculated in 6-well plates. When the growth area reached about 80% on the second day, the cells were cultured in complete medium containing different final concentrations of fumaric acid (0, 0.1, 0.2, 0.4, 0.6, 0.8 mg / mL) for 4 h. 0 mg / mL was DMSO dissolved in fumaric acid, which served 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 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 HiScript II 1st Strand cDNA Synthesis Kit (Vazyme, China). Then, the expression levels of GCRV 873S6 gene and interferon system-related genes (IFN1, IFN3, IRF3 and IRF7) were detected by qPCR. 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). Three replicates were set for each sample, and the β-actin gene was used as an internal reference to normalize the expression of the gene. The primer sequences for amplifying these genes are shown in Table 1. The relative expression levels of the genes were measured using 2 -△△Ct Method calculation.

[0050] Table 1 Primer sequences for qPCR

[0051]

[0052]

[0053] 3) Analysis of GCRV infection results and gene expression of immune-related factors

[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 concentration of fumaric acid (0.6 mg / mL, 0.8 mg / mL) can simultaneously induce the up-regulation of IFN1, IFN3, IRF3 and IRF7 gene expression, among which IFN1 and IRF7 expression levels were higher, while low concentration of fumaric acid (0.1 mg / mL, 0.2 mg / mL) inhibited the expression of IFN and IRF, which was basically opposite to the gene expression changes of GCRV 873 in cells, once again indicating 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 showed significant anti-GCRV 873 virus function in in vitro experiments, and was significantly affected by its concentration. High concentrations of fumaric acid (0.6mg / mL, 0.8mg / mL) can 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 expression changes of IFN1, IFN3, IRF3 and IRF7 genes showed a basically opposite trend to the changes in the GCRV873 virus content, which further supports that fumaric acid may exert its antiviral effect through immune enhancement.

[0058] Example 3 Application of fumaric acid in antiviral preparations

[0059] 1. Artificial infection and virus attack experiment

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

[0061] In an indoor cement pool, 24 cages were configured to divide the same water body into 24 spaces, and 50 grass carps (13±4cm, 35±11g) were placed in each cage. The fish were cultured in flowing water and filled with water to ensure sufficient dissolved oxygen. The 24 cages were set up as normal feed group, fumaric acid group (the amount of fumaric acid used was 0.5%, 1% and 2% of the feed weight), astragalus polysaccharide group (the amount of astragalus polysaccharide used was 0.1% and 1% of the feed weight) and five yellow powder group (the amount of five yellow powder used was 0.5% and 1% of the feed weight), and each treatment concentration was paralleled 3 times. Astragalus polysaccharide and five yellow powder were two conventional feed enhancement additives, respectively used as two groups of positive drug controls, and the feed used was 2-3mm specification 30% protein content expanded granular feed, purchased from Tongwei Co., Ltd. The grass carps of the four types of treatments were fed continuously for 14 days according to the feed group, fed once in the morning and evening, and the weight of the feed fed every day was about 2% of the fish 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 infection, 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%, 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 findings Figure 4 As shown, after adding fumaric acid to the feed, the damage of grass carp tissues infected with GCRV was significantly reduced, and the degree of nuclear condensation 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 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. Experiments to simulate natural breeding conditions

[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 put into the ponds in June 2023, and then the water temperature was monitored every day. When the water temperature exceeded 27°C, fumaric acid was added to the feed at 1% of the weight of the feed and then fed to the fish for 14 consecutive days in the morning and evening. As of December 2023, there was no outbreak of hemorrhagic disease in the grass carp cultured in the three ponds. The results show 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 protection scope 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.

4. The use according to claim 2 or 3, characterized in that The formulation comprises fish feed.

5. The use according to claim 4, characterized in that The feed is dry granular feed or fermented liquid feed.

6. The use according to claim 4, characterized in that The added amount of fumaric acid in the feed is 1wt% of the feed.

7. Use of fumaric acid in the preparation of a drug for inhibiting the replication of grass carp reovirus in cells.

8. Use of fumaric acid in the preparation of an immunopotentiator for grass carp reovirus-infected cells.

9. The use according to any one of claims 3, 7 and 8, characterized in that In the culture medium of the cells, the added amount of fumaric acid is 0.6-0.8 mg / mL.

10. The use according to any one of claims 1-3 and 7-8, 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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