CN119157868B - Application of fumaric acid in preventing and treating grass carp reovirus infection - Google Patents
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Abstract
The invention relates to the field of aquaculture. The invention discloses a new application of fumaric acid in preparing a medicament for preventing and treating Grass Carp Reovirus (GCRV) infection. The research of the invention shows that fumaric acid has remarkable antiviral efficacy on GCRV in-vitro experiments and in-vivo experiments. Fumaric acid in cell culture system can inhibit the duplication of GCRV, reduce viral load obviously, promote the expression of immune related factor and strengthen the immune defense mechanism of host cell. In addition, the artificial infection toxicity attack experiment proves that the average survival rate of the grass carp population of the fumaric acid treatment group can be obviously improved by 19.60 percent compared with that of the grass carp population of the untreated group when the grass carp population faces GCRV infection. In an experiment simulating a natural culture state, the periodic feeding of the feed containing fumaric acid successfully prevents the outbreak of grass carp hemorrhagic disease. The invention firstly explores the application potential of the fumaric acid in the fields of antivirus and immunity enhancement, and opens up a new way for the multifunctional application of the fumaric acid in the field of aquaculture.
Description
Technical Field
The invention relates to the field of aquaculture, in particular to application of fumaric acid in preventing and treating grass carp reovirus infection.
Background
At present, the grass carp cultivation amount in China accounts for about 20% of the total cultivation amount, and the annual yield reduction caused by diseases accounts for more than 30% of the total annual grass carp cultivation amount. Among grass carp pathogens, grass carp reovirus (GRASS CARP reovirus, GCRV) is the most harmful, and grass carp hemorrhagic disease caused by infection has high infectivity and high lethality, and causes large-scale yield reduction, so that the grass carp hemorrhagic disease is always considered as one of the most prominent problems in freshwater aquaculture in China. The grass carp reovirus particles are icosahedral and symmetrical, have double-layer capsids and have no envelope, the genome consists of 11 double-stranded RNAs, and the grass carp reovirus infection can cause congestion and bleeding phenomena of different degrees of organs and tissues of grass carp, and the morbidity and mortality rate is up to more than 80%. At present, no ideal treatment method for grass carp reovirus infection exists, and the prevention work is mainly adopted. Grass carp hemorrhagic disease caused by grass carp reovirus is a major viral disease that jeopardizes the grass carp farming industry. Grass carp reovirus is capable of in vitro culture in a variety of grass carp cell lines, such as grass carp kidney Cells (CIK), grass carp fin Cells (CF), grass carp ovary cells (GCO), and Grass Carp Blasts (GCB), and produces significant cytopathic effects.
At present, the prevention and treatment mode aiming at GCRV is mainly focused on vaccine research. With the development of genetic engineering vaccines, various grass carp hemorrhagic disease vaccines represented by subunit vaccines and nucleic acid vaccines appear successively. Currently, various types of vaccines are mainly used by injection, oral administration or bathing. However, the mass production and application of vaccines are still limited, mainly because the strong specificity of vaccines makes them unusable for viruses that are prone to variation. Based on the above, the anti-GCRV drug with good antiviral effect is found to have remarkable advantages compared with vaccine control in the aspects of maintaining antiviral effect of different strains. At present, no specific medicine aiming at grass carp hemorrhagic disease exists, even the early common hemorrhagic disease prevention and control medicines such as ribavirin also have cancerogenic side effects, and the prevention and control medicines are forbidden, so that the screening of a medicine for preventing and treating GCRV infection with low side effects is very important.
Disclosure of Invention
In view of this, the present invention performed DIA proteomic and non-targeted metabonomic analyses of five tissues, gill, liver, intestine, kidney and muscle, before and after GCRV infection, and found that carbohydrate metabolism was the metabolic pathway that was primarily affected after GCRV infection. Further studies on the metabolic pathways of butyric acid belonging to carbohydrate metabolism in kidney tissues were then carried out, and the metabolic intermediate fumaric acid was found to have antiviral effects. Based on the research on feasibility of fumaric acid in preventing and treating GCRV infection, the invention discovers that fumaric acid has a remarkable effect of inhibiting GCRV replication and can induce cell immunity enhancement. In addition, the invention also discovers that the fumaric acid can obviously improve the survival rate of the juvenile grass carp infected with GCRV.
The technical scheme of the invention is realized as follows:
in a first aspect, the invention provides the use of fumaric acid in the manufacture of a medicament for the treatment of a grass carp reovirus infection.
In a second aspect, the invention provides the use of fumaric acid in the preparation of a formulation for the control of grass carp reovirus infection.
In a third aspect, the invention provides the use of fumaric acid in the preparation of a formulation for inhibiting replication of grass carp reovirus in a cell.
In some preferred embodiments, the formulation comprises fish feed. The feed is dry granular feed or liquid feed after fermentation treatment.
In some preferred embodiments, the fumaric acid is added to the feed in an amount of 1wt% of the feed.
In a fourth aspect, the invention provides the use of fumaric acid in the manufacture of a medicament for inhibiting replication of grass carp reovirus in a cell.
In a fifth aspect, the invention provides the use of fumaric acid in the manufacture of an immunopotentiator for grass carp reovirus infected cells.
In some preferred embodiments, the fumaric acid is added in an amount of 0.6-0.8mg/mL in the medium of the cells.
Further, the grass carp reovirus includes at least one of a type I strain and a type II strain.
The beneficial effects of the invention at least comprise the following:
The invention adopts independent data acquisition (DIA) proteomics and non-targeted metabonomics to carry out two kinds of histology sequencing on virus main dip-dyed tissue samples, and combines biological information analysis and biological statistics analysis of data to identify metabolite molecules which are extremely remarkably down-regulated and expressed, thereby screening and obtaining candidate GCRV prevention and treatment preparations or medicaments, namely fumaric acid.
The invention discloses for the first time that fumaric acid has remarkable antiviral effect on GCRV on a cell-individual level. Specifically, fumaric acid shows excellent antiviral activity in a cell culture system within the concentration range of 0.6-0.8mg/mL, so that the replication of GCRV is effectively inhibited, the viral load is obviously reduced, the expression of immune related factors is promoted, the immune defense mechanism of host cells is enhanced, and a brand new view and thought are provided for prevention and control strategies for viral diseases in the aquaculture industry.
The invention also proves the antiviral effect of fumaric acid in the actual culture environment through an artificial infection challenge experiment, namely, when the grass carp population of the fumaric acid treatment group faces GCRV infection, the average survival rate of the grass carp population can be obviously improved by 19.60 percent compared with that of the grass carp population of the untreated group, and the result directly and forcefully proves the effectiveness of fumaric acid in improving the antiviral capability and the culture survival rate of fishes.
In addition, in the experiment simulating the natural culture state, the periodic feeding of the feed containing fumaric acid successfully prevents the outbreak of grass carp hemorrhagic disease, and further proves the stability and the practicability of the grass carp hemorrhagic disease in a complex ecological environment. Fumaric acid, which is a widely accepted feed additive, has been well documented in terms of its safety and functionality in promoting fish growth. The invention discloses the potential value of the fumaric acid in the antiviral field for the first time, and the recommended use amount (1 wt%) is in the known safety range (0.5-2 wt%) without negative influence on the water environment, thus opening up a new way for the multifunctional application of the fumaric acid in aquaculture.
Therefore, in view of the remarkable effects of fumaric acid in inhibiting viral infection and enhancing organism immunity, the invention is expected to provide a new method for preventing and treating viral diseases or other diseases in aquaculture. Fumaric acid is used as a naturally occurring organic acid, and also meets the requirements of green cultivation technology. And simultaneously combines the dual functions of the fumaric acid in the aspects of promoting growth and resisting viruses, and can be developed into a multifunctional feed additive in the future, thereby providing a more comprehensive and efficient solution for the aquaculture industry.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, it being obvious that the drawings described below are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the results of detecting the expression of GCRV 873S6 gene under different concentrations of fumaric acid in the examples of the present invention;
FIG. 2 shows the results of detection of gene expression of immune-related factors (IFN 1 and IFN3, members of the interferon family, shown in panel A, and IRF3 and IRF7, respectively) by different concentrations of fumaric acid treatment in the examples of the present invention;
FIG. 3 shows the survival statistics of fumaric acid group, positive drug group (Astragalus polysaccharides group, five yellow powder group) and control group (blank control) of GCRV artificial infection challenge experiment in the embodiment of the present invention;
FIG. 4 shows the results of liver, spleen and kidney histopathological examination of GCRV in an embodiment of the present invention in an artificial infection challenge experiment.
Detailed Description
In order to make the objects, technical solutions and advantages of the present application more apparent, the present application will be described in further detail with reference to the following examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the application. The reagents which are not specifically described in the present application are conventional reagents and are commercially available, and the methods which are not specifically described in the present application are conventional experimental methods and are known from the prior art.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The terms "comprising" and "having" and any variations thereof, are intended to cover a non-exclusive inclusion.
EXAMPLE 1 drug screening for the control of GCRV infection
1. Proteome and metabolome sequencing
1) Acquisition and processing of experimental groupings and sample organizations
As an experimental group, 1 identical cell population (15+ -3 cm, 40+ -10 g,100 tails) of June-aged grass carp was subjected to GCRVHZ stomach-lavage and virus-challenge, the virus copy number was 7.42×10 8 copies/mL, and the stomach-lavage dose was 500. Mu.L/tail. After the toxicity is removed, samples are collected when obvious symptoms such as outlier single swimming, swimming unbalance, body surface bleeding and the like appear on grass carp after virus infection, and 5 tissues of gill, liver, intestine, kidney and muscle are respectively collected from each fish. 100 healthy grass carp of the same genetic background served as a 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 are mixed into one sample to be sequenced, three parallel samples are arranged for each sample to be sequenced, and 30 samples (5 multiplied by 3 multiplied by 2) of the experimental group and the control group are obtained.
2) Proteome sequencing and analysis
DIA proteome liquid phase separation of 30 samples was performed on a high performance liquid chromatograph UltiMate UHPLC (Thermo FISHER SCIENTIFIC, san Jose, calif.) and a mass spectrometer Orbitrap Exploris (Thermo FISHER SCIENTIFIC, SANJOSE, CA) using Shimadzu LC-20AD liquid phase system (Shimadzu, japan) DDA library construction and DIA sample analysis. DDA data were pooled using MaxQuant software (version 1.5.3.30) as a spectral library for DIA analysis. Spectronaut software was used to analyze the DIA data and iRT peptide fragments were used to correct for retention time. False positive control was then completed at FDR 1% based on the Target-decoy model applicable by SWATH-MS. Error correction and normalization were performed for each sample, and significant differential protein screening was performed according to Fold Change (FC). Gtoreq.2 and P < 0.05.
The results showed that 30 samples quantified 114015 peptide fragments and 10808 proteins in total, with significantly more up-regulated protein than down-regulated protein in gill, liver, kidney and muscle tissue, and more down-regulated protein in the intestine, with the most differentially expressed protein in the kidney, with 1529 up-regulated proteins and 219 down-regulated proteins.
3) Metabolome sequencing and analysis
Non-targeted metabolome UPLC-MS analysis of 30 samples metabolite separation and detection was performed in ESI mode using Waters 2777C UPLC (Waters, USA) tandem Q Exactive HF high resolution mass spectrometer (Thermo FISHER SCIENTIFIC, USA). And importing CompoundDiscoverer 3.3.3 (Thermo FISHER SCIENTIFIC, USA) of mass spectrometry off-line data into 3835.3 (Thermo FISHER SCIENTIFIC, USA) software, and carrying out mass spectrometry data analysis by combining a BMDB database, a mzCloud database and a CHEMSPIDER online database to obtain a data matrix containing information such as metabolite peak area, identification result and the like. And then importing CompoundDiscoverer derived results into metaX for data preprocessing, normalizing the data by a probability quotient normalization method (probabilic QuotientNormalization, PQN) to obtain relative peak areas, correcting batch effects by QC-RLSC (Quality control-based robustLOESS signal correction) and partial polynomial regression fit signal correction, and finally deleting compounds with relative peak areas CV (Coefficient ofVariation and variation coefficients) of more than 30% in all QC samples. And calculating the P value by utilizing univariate analysis to calculate Fold change and t-test, and screening the remarkably different metabolites according to FC not less than 1.2 or not more than 0.83 and P < 0.05.
As a result, 4040 metabolites were identified in total, and the number of metabolites up-and down-regulated was 646 and 875, respectively, as the same as the number of metabolites differentially expressed by kidneys.
2. Screening of metabolic molecules in critical pathways
1) KEGG enrichment analysis and common pathway identification
KEGG enrichment analysis was performed on DEP and DEM of each tissue at the protein and metabolite levels, respectively, and pathways with P <0.05 were selected to intersect to obtain a common pathway for each tissue at the protein and metabolite levels.
The result was a total of 16 pathways, carbohydrate metabolism (6) and some immune related amino acid metabolism (4), and also lipid metabolism (2), prosthetic and vitamin metabolism (2), energy metabolism (1) and transport catabolism (1), respectively. The results indicate that GCRV infection primarily affects host carbohydrate metabolic pathway changes.
2) Deep analysis of kidney differential metabolic pathways
The kidneys with the largest difference proteins and metabolites were selected for further analysis, and only butyric acid metabolism was attributed to carbohydrate metabolism in the different metabolic pathways of the kidneys, and total of 14 DEPs and DEMs were enriched in the butyric acid metabolic pathways, 7 of which were concentrated in the tricarboxylic acid cycle. For example, 4-aminobutyric acid aminotransferase (fc=2.43, p < 0.05) and glutamate decarboxylase 1, which promote TC a cycle, were up-regulated (fc=4.87, p < 0.0001), alpha-ketoglutarate (fc=7.04, p < 0.001) and succinic acid (fc=1.48, p < 0.05), fumaric acid (fc=0.55, p < 0.0001), maleic acid (fc=0.54, p < 0.01) and pyruvic acid (fc=0.47, p < 0.05), which are intermediates of TCA cycle, were down-regulated.
3. Screening results the proteome and metabolome data of the present invention show that fumaric acid is down-regulated in renal butyrate metabolism (p=7.33x10 -5) at GCRV infection, and that the P-value of fumaric acid is significantly lower and the reliability is higher compared to other metabolites such as maleic acid (p=1.09 x 10 -3) and pyruvic acid (p=1.41 x 10 -2), thus fumaric acid is selected as a candidate drug for preventing GCRV infection.
EXAMPLE 2 use of fumaric acid in cells for antiviral and immunopotentiating functions
1) Cell culture, fumaric acid treatment and virus attack
Fumaric acid (product number: S30198-500 g) was purchased from Shanghai Yeast Biotechnology Co., ltd, CIK cells (grass carp kidney cells) were inoculated into a 6-well plate, and when the growth area of the next day reached about 80%, complete medium culture cells containing fumaric acid (0, 0.1,0.2,0.4,0.6,0.8 mg/mL) at different final concentrations were replaced for 4 hours, and 0mg/mL was DMSO dissolved in fumaric acid as a control group. After 4h, 100. Mu.L of GCR V873 virus solution (3.76X10 7 copies/mL) was added to attack the virus, and after 24h the virus was removed, the cells were collected.
2) RNA extraction and Gene expression detection
The collected cell samples were all RNA extracted by Trizol (FISHER SCIENTIFIC, USA) method and reverse transcription was performed using HISCRIPT II 1st Strand cDNA Synthesis Kit kit (Vazyme, china). Then, the expression amounts of the GCRV 873S6 gene and the interferon system related genes (IFN 1, IFN3, IRF3 and IRF 7) were detected by qPCR method, the reaction system was configured using HISCRIPT IIQ RT SuperMix for qPCR + GDNA WIPER (Vazyme, china), the PCR reaction was carried out in a real-time fluorescent quantitative PCR apparatus (CF X96, bio Rad), 3 replicates were set for each sample, and the beta-actin gene was used as the expression of the internal reference normalized gene. The primer sequences for amplifying the genes are shown in Table 1, and the relative expression amount of the genes is calculated by adopting a2 -△△Ct method.
TABLE 1 primer sequences for qPCR
3) GCRV infection results and immune related factor gene expression analysis
As shown in fig. 1, qPCR results showed that the content of GCRV 873 in the high concentration fumaric acid treated group (0.6 mg/mL, 0.8 mg/mL) was significantly reduced (P < 0.0001) compared to the control group 24h after GCRV 873 infection, while the content of GCRV 873 in the low concentration fumaric acid treated group (0.1 mg/mL, 0.2mg/mL, 0.4 mg/mL) was not significantly changed, indicating that high concentration fumaric acid had the function of anti-GCRV in vitro experiments.
As a result, as shown in FIG. 2, high concentration of fumaric acid (0.6 mg/mL, 0.8 mg/mL) can induce up-regulation of the expression levels of IFN1, IFN3, IRF3 and IRF7 genes simultaneously, wherein the up-regulation of the expression levels of IFN1 and IRF7 is higher, while low concentration of fumaric acid (0.1 mg/mL, 0.2 mg/mL) inhibits the expression of IFN and IRF, which is a substantially opposite trend to the change of the gene expression of GCRV 873 in cells, again demonstrating that fumaric acid concentration is the key to inhibit GCRV infection, and that this effect may be mediated by the interferon system.
4) Conclusion(s)
Fumaric acid has obvious function of resisting GCRV 873 virus in-vitro experiments, is obviously influenced by the concentration of the fumaric acid, and high-concentration fumaric acid (0.6 mg/mL and 0.8 mg/mL) can obviously inhibit the replication of the virus. In addition, fumaric acid can exert antiviral effect through immune enhancement, and high-concentration fumaric acid can induce up-regulation of expression of genes such as IFN1, IFN3, IRF7 and the like, so that antiviral capability of cells is enhanced. The expression changes of IFN1, IFN3, IRF3 and IRF7 genes are in a basically opposite trend to the change of the GCRV 873 virus content, and further support that fumaric acid can play an antiviral effect through immune enhancement.
EXAMPLE 3 use of fumaric acid in antiviral Agents
1. Experiment for eliminating toxicity of artificial infection
1) Experiment grouping, feed additive configuration and feeding management
In an indoor cement pond, 24 net cages are arranged to divide the same water body into 24 spaces, 50 grass carps (13+/-4 cm, 35+/-11 g) are placed in each net cage, and the water body is cultivated in a running way and is filled with oxygen to ensure sufficient dissolved oxygen. The 24 net cages are respectively arranged into a normal feed group, a fumaric acid group (the fumaric acid usage amount is 0.5%, 1% and 2% of the feed weight), an astragalus polysaccharide group (the astragalus polysaccharide usage amount is 0.1% and 1% of the feed weight) and a five-yellow powder group (the five-yellow powder usage amount is 0.5% and 1% of the feed weight), each treatment concentration is 3 parallels, the astragalus polysaccharide and the five-yellow powder are two conventional feed enhancement additives, and are respectively used as two groups of positive drug controls, the used feed is an expanded pellet feed with the protein content of 30% in the specification of 2-3mm, and the expanded pellet feed is purchased from Tongwei Co. The grass carp treated in 4 classes is continuously fed for 14 days according to the feed group, 1 time in the morning and at night, and the weight of the fed feed is about 2% of the weight of the fish every day.
2) Artificial infection virus attack, histopathological detection and survival rate statistics
Each fish was perfused with GCRVHZ. Mu.L of virus liquid (7.42X10. 10 8 copies/mL) after 14 days, the death status of each group of fish was recorded every day after the challenge, and normal feed group and fumaric acid group (1%) of diseased fish were collected and liver, spleen and kidney tissues were taken for pathological section observation. As shown in fig. 3, the average survival rate of the normal feed group was 4.08%, the survival rates of the fumaric acid group (0.5%, 1% and 2%) were 10.00%, 23.68% and 20.67%, the survival rates of the astragalus polysaccharide group (0.1% and 1%) were 7.33% and 6.67%, and the average survival rates of the pentraxin group (0.5% and 1%) were 9.33% and 8.67%, respectively. The survival rate of the fumaric acid group (1%) is improved by 14.35-19.60 percent compared with the control group and the positive drug group.
Histopathological observations as shown in fig. 4, after fumaric acid was added to the feed, the damage of grass carp tissue infected with GCRV was significantly reduced, and the degree of nuclear shrinkage and cell gap in the liver, the cell gap in the spleen, and the degree of tubular shrinkage and monocyte number in the kidney were all smaller than those of the normal feed group.
3) Compared with a normal feed group, the average survival rate of the grass carp in the fumaric acid group is obviously improved (from 4.08% to 23.68%), which shows that fumaric acid plays an important role in protecting the grass carp against GCRV infection, and the survival ability of the fish can be obviously improved. Histopathological observations further confirm the protective effect of fumaric acid. In the fumaric acid group, the damage of key organs such as liver, spleen, kidney and the like caused by GCRV infection is obviously reduced, which indicates that fumaric acid can relieve the damage of viruses to fish tissues. Compared with astragalus polysaccharide and five-yellow meal, the fumaric acid group has higher survival rate and shows the superiority in improving the antiviral ability of grass carp.
2. Experiment for simulating natural culture state
3 Outdoor ponds (1 mu, 1 mu and 2.5 mu) with bleeding diseases of grass carp in 2022 are sequentially put into young grass carp of January age (2000 tails/mu) in 2023 and 6 months, then water temperature is monitored every day, and when the water temperature exceeds 27 ℃, fumaric acid is added according to 1% of the weight of feed for mixing, feeding is carried out continuously for 14 days in the morning and evening. No grass carp cultured in 3 ponds of 12 months of 2023 has outbreak hemorrhagic disease. The result shows that the grass carp hemorrhagic disease outbreak can be effectively prevented by feeding the feed with fumaric acid.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (6)
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