Application of AGPAT gene as a target in anti-infectious hematopoietic necrosis virus

Abstract

The application discloses application of AGPAT gene as a target point in anti-infectious hematopoietic organ necrosis virus. The application provides application of a substance capable of inhibiting AGPAT gene expression in preparation of a product for resisting the infectious hematopoietic organ necrosis virus. The application provides application of rainbow trout AGPAT in preparation of a medicine for preventing or treating IHNV infection, provides a new target point and treatment scheme for preventing and treating occurrence and development of the IHNV infection disease, and has important significance for effectively improving the prevention and control level of the rainbow trout virus disease.

Description

Technical Field

[0001] This invention relates to the field of aquaculture, specifically to the application of the AGPAT gene as a target in the treatment of infectious hematopoietic necrosis virus. Background Technology

[0002] Infectious hematopoietic necrosis virus (IHNV) is a highly contagious pathogen in salmonid fish, causing mortality rates exceeding 90% in juvenile rainbow trout. This virus is a single-stranded, negative-sense RNA virus with a genome of approximately 11 kb, encoding six proteins: nucleocapsid (N), polymerase-associated phosphorylated protein (P), matrix protein (M), surface glycoprotein (G), nonviral particle protein (NV), and viral RNA polymerase (L). As a typical enveloped virus, IHNV requires a large amount of lipids in the host cell to provide sufficient energy and materials for viral replication. Phospholipids, mainly including phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and phosphatidylinositol, are important components of the lipid bilayer. Existing research has shown that rotavirus, dengue virus, hepatitis B virus, poliovirus, grass carp reovirus, coronavirus, enterovirus, influenza virus, etc., can all reprogram lipid metabolism processes by regulating lipid metabolism enzymes in host cells in order to complete their own replication and proliferation.

[0003] 1-Acylglycerol-3-phosphate-O-acyltransferase (AGPAT) is one of the rate-limiting enzymes in the glycerol lipid synthesis pathway. AGPAT primarily regulates the de novo synthesis of phosphatidic acid (PA) and lysophosphatidic acid (LPA) by acylation. The de novo PA then undergoes a series of acylation reactions to form diacylglycerol (DAG) and triacylglycerol (TAG). Simultaneously, DAG can further react with CDP-choline or CDP-ethanolamine in the endoplasmic reticulum to form the corresponding poly(PC) or poly(PE). Additionally, PA can be converted to CDP-DAG by CDP-diaacylglycerol synthase (CDS), leading to the derivation of two other important membrane lipid components: phosphatidylglycerol (PG) and phosphatidylinositol (PI). Therefore, AGPAT participates in various biological processes in vivo, playing a crucial role in regulating intracellular phospholipid metabolism, maintaining membrane stability and permeability, and signal transduction, among other physiological and pathological processes. However, there are relatively few studies on the differential changes in host cell lipid metabolites after IHNV infection, and the analysis of key genes (such as AGPAT) that regulate related differential lipid metabolites is not very clear. Therefore, clarifying how the virus causes changes in host cell lipids to meet its own replication and proliferation needs is essential for revealing the mechanism of virus-host interaction, which also provides a new target for the prevention and treatment of IHNV infection. Summary of the Invention

[0004] The purpose of this invention is to provide the application of the AGPAT gene as a target in the treatment of infectious hematopoietic organ necrosis virus (IHNV).

[0005] In a first aspect, the present invention claims the use of a substance capable of inhibiting AGPAT gene expression in the preparation of products for use against infectious hematopoietic organ necrosis virus (IHNV).

[0006] Secondly, the present invention claims the use of a substance capable of inhibiting AGPAT gene expression in the preparation of products for the prevention and / or treatment of diseases caused by infectious hematopoietic organ necrosis virus (IHNV) infection.

[0007] Furthermore, the disease is infectious hematopoietic necrosis.

[0008] Thirdly, the present invention claims protection for the use of a substance capable of inhibiting AGPAT gene expression in any of the following:

[0009] (A1) Prepare products for inhibiting the replication of infectious hematopoietic organ necrosis virus (IHNV) in host cells;

[0010] (A2) Prepare a product for reducing the titer of infectious hematopoietic organ necrosis virus (IHNV) at the cellular level.

[0011] In all of the above aspects, the product may be a medicine or a vaccine.

[0012] Fourthly, the present invention claims the use of substances capable of inhibiting AGPAT gene expression in the preparation of cell or animal models that enhance resistance to infectious hematopoietic organ necrosis virus (IHNV).

[0013] In the above aspects, the substance that can inhibit AGPAT gene expression can be a substance that directly targets the AGPAT gene and can reduce AGPAT gene expression.

[0014] Furthermore, the substance capable of inhibiting AGPAT gene expression can be siRNA targeting the AGPAT gene. In a specific embodiment of the present invention, the siRNA targeting the AGPAT gene is a double-stranded RNA formed by SEQ ID No. 1 and SEQ ID No. 2.

[0015] Fifthly, the present invention claims a method for preparing a cell model or animal model with enhanced resistance to infectious hematopoietic organ necrosis virus (IHNV).

[0016] The method for preparing a cell model or animal model with enhanced resistance to infectious hematopoietic organ necrosis virus (IHNV) claimed in this invention may include the following steps: reducing the expression of the AGPAT gene in host cells or animals to obtain recombinant cells or animals; and the recombinant cells or animals exhibiting enhanced resistance to infectious hematopoietic organ necrosis virus (IHNV) compared to the host cells or animals.

[0017] Furthermore, reducing the expression of the AGPAT gene in the host cells or animals can be achieved by introducing siRNA targeting the AGPAT gene into the host cells or animals.

[0018] In a specific embodiment of the present invention, the siRNA targeting the AGPAT gene is a double-stranded RNA formed by SEQ ID No. 1 and SEQ ID No. 2.

[0019] Sixthly, the present invention claims protection for any of the following applications:

[0020] (B1) Application of the AGPAT gene as a target in regulating the resistance of host cells or animals to infectious hematopoietic necrosis virus (IHNV).

[0021] Furthermore, the application is not for disease diagnosis or treatment.

[0022] (B2) Application of AGPAT gene as a target in regulating the replication ability of infectious hematopoietic necrosis virus (IHNV) in host cells or animals;

[0023] Furthermore, the application is not for disease diagnosis or treatment.

[0024] (B3) Application of the AGPAT gene as a target in the preparation of products for regulating the resistance of host cells or animals to infectious hematopoietic necrosis virus (IHNV).

[0025] (B4) The application of the AGPAT gene as a target in the preparation of products for regulating the replication ability of infectious hematopoietic necrosis virus (IHNV) in host cells or animals.

[0026] Seventhly, the present invention claims protection for cell models prepared using the method described in the sixth aspect above.

[0027] In all of the above aspects, the cells may be cells capable of being infected by infectious hematopoietic organ necrosis virus (IHNV), such as fish cells.

[0028] In a specific embodiment of the present invention, the fish cells are RTG-2 cells.

[0029] In all the above aspects, the animals mentioned are animals that can be infected with infectious hematopoietic necrosis virus (IHNV), such as fish (e.g., rainbow trout).

[0030] In all the above aspects, the nucleotide sequence of the AGPAT gene is shown in SEQ ID No. 3.

[0031] This invention provides the application of the AGPAT gene in the preparation of drugs for the prevention or treatment of IHNV infection, providing a new target and treatment plan for the prevention and treatment of IHNV infection, which is of great significance for effectively improving the prevention and control of rainbow trout virus disease. Attached Figure Description

[0032] Figure 1 The effects of IHNV infection on changes in lipid metabolites in host cells are shown in Figure A. A shows the effect of IHNV infection on changes in the types of lipid metabolites in host cells; B shows the principal component analysis plot of changes in lipid metabolites in host cells caused by IHNV infection; C shows the partial least squares discriminant analysis plot of changes in lipid metabolites in host cells caused by IHNV infection.

[0033] Figure 2This section illustrates the effect of IHNV infection on changes in the content of lipid metabolites in host cells. A is a volcano plot showing the differences in lipid metabolism in host cells caused by IHNV infection; B is a heatmap showing the differences in lipid metabolism in host cells caused by IHNV infection; C is the effect of IHNV infection on the content of phospholipid metabolites in host cells.

[0034] Figure 3 This study focuses on the enrichment analysis of differentially expressed lipid metabolism products and the corresponding differentially expressed regulatory genes. A represents the enrichment analysis of differentially expressed lipid metabolites in host cells induced by IHNV infection; B represents the top 20 differentially expressed genes (TOP20) regulating corresponding lipid metabolism in host cells induced by IHNV infection; and C represents the validation analysis of the differentially expressed genes.

[0035] Figure 4 The changes in AGPAT gene expression during IHNV infection and the effect of AGPAT gene silencing on viral replication are shown in Figure A. Figure B shows the change in AGPAT gene expression during IHNV infection. Detailed Implementation

[0036] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0037] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0038] Example 1: Application of the AGPAT gene as a target in the treatment of infectious hematopoietic necrosis virus.

[0039] I. Materials and Methods

[0040] 1. Cells and viruses

[0041] Rainbow trout gonadal fibroblasts (RTG-2 cells) were preserved in complete culture medium (basal medium supplemented with 10% fetal bovine serum (FBS) (Gibco BRL, Grand Island, NY, USA), 100 units / mL penicillin and streptomycin) at 15°C and 5% CO2.

[0042] The IHNV-Sn1203 strain (Genbank accession number: KC660147.1) is stored in the applicant's laboratory.

[0043] 2. Viral infection

[0044] With a density of 1×10 5 RTG-2 cells were seeded in 6-well plates and cultured overnight in complete medium. Cells were then infected with the IHNV-Sn1203 virus strain with an MOI of 1.0 and incubated at 15°C and 5% CO2 to establish an IHNV challenge model. Uninfected cells served as a simulated control. Cells from the IHNV challenge group (IH group) and the simulated control group (MC group) were collected 72 h after infection.

[0045] 3. Preparation and analysis of lipidomics samples

[0046] Infected and uninfected IHNV-positive RTG-2 cells were collected, washed with frozen PBS buffer, and lysed using an ice-bath sonication homogenizer. Cellular lipids were extracted using a methanol / chloroform (1 / 2, v / v) solution. The solution was then dried in a freeze-drying centrifuge and resuspended in isopropanol:methanol (1 / 1, v / v). After centrifugation, the supernatant was analyzed for lipid metabolites by BGI Genomics Co., Ltd. (Shenzhen, China). All samples were mixed in equal volumes for quality control (QC).

[0047] The raw LC-MS data were processed using progenesis QI software (Version 2.3, Waters Corporation, Milford, USA) for feature alignment, non-target signal detection, and signal integration to obtain lipidomics data including m / z, retention time, and peak intensity. Principal component analysis (PCA) and (orthogonal) partial least squares discriminant analysis (PLS-DA) were used to model changes after mean centering and Pareto variance scaling, respectively. Variables with VIP values ​​(VIP>1) were used as the initial screening standard for differentially expressed lipid molecules among groups. Then, univariate analysis with t-tests (P<0.05) and fold change analysis (FC>1.2 or <0.5) was used to further identify differentially expressed lipid molecules. Cellular pathway analysis relied on the KEGG database (http: / / www.genome.jp / kegg / ) and lipid maps (http: / / www.lipidmaps.org / ).

[0048] 4. RNA-seq data analysis

[0049] The expression levels of lipid metabolism-related genes were derived from our previous analysis (Comparative transcriptome analysis of long non-coding RNA (lncRNA) in RTG-2 cells infected by infectious hematopoietic necrosis virus, Ren G. et al., Fish and Shellfish Immunology, 2022). Differentially expressed transcripts between different samples or groups were analyzed using DEGseq software (log2FC|>2, p<0.05). The identified differentially expressed genes (DEGs) were further clustered, and KEGG enrichment analysis was performed using the Pheatmap and Phyper packages in r.

[0050] 5. RT-qPCR analysis

[0051] RNA was extracted according to the instructions, and its quality was measured using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, USA). RT-qPCR experiments were performed using an Applied Biosystems 7500 Real-Time PCR system (Bio-Rad, Hercules, CA, USA) to detect IHNV-N and glycerophospholipid (GP) metabolism-related genes, and to verify changes in eight selected DEGs. Primers are shown in Table 1. Using β-actin as an internal control, the relative expression levels of gene transcripts (22) were calculated using the comparative cycle threshold (Ct) method. -ΔΔCt ).

[0052] Table 1. RT-qPCR primers

[0053]

[0054]

[0055] 6. AGPAT-RNA interference (RNAi)

[0056] The nucleotide sequence of the AGPAT gene is shown in SEQ ID No. 3.

[0057] Specific siRNA oligonucleotides targeting the AGPAT gene (SEQ ID No. 1: 5'-GGAGUCCGUACCAGAAGAUTT-3'; SEQ ID No. 2: 5'-AUCU UCUGGUACGGACUCCTT-3') were obtained from GenePharma (Shanghai, China). RTG-2 cells were seeded in 6-well plates to ensure 70-80% coverage. Appropriate volumes of RNAiMAX reagent (Thermo Fisher Scientific, USA) and primer solutions were added to Opti-MEM medium and mixed thoroughly at room temperature for 5 min. Subsequently, the siRNA mixture or negative control (treatment group treated with transfection reagent only) was transfected into cells for 24 h, followed by incubation with IHNV-Sn1203 (MOI = 0.1) for 24 h, 36 h, 48 h, and 72 h. Cells were collected and total RNA was extracted for subsequent detection. Viral titers were determined using TCID50. 50 The method was used for determination.

[0058] II. Results and Analysis

[0059] 1. Overview of cellular lipidomics analysis 72 hours after IHNV infection

[0060] The results show that the lipid metabolites identified in IHNV-infected RTG-2 cells mainly belong to 6 lipid classes and 35 subclasses, totaling 805 lipid metabolites. Figure 1 (A). Principal component analysis (PCA) results showed high reproducibility and variability between the MC and IH groups. Figure 1 Partial least squares regression analysis (PLS-DA) showed that (R... 2 (Y = 0.99, Q2 = 0.88) Significant differences were observed in changes in lipid metabolites after viral infection. Figure 1 (C) Glycerolphospholipids were the category that caused the most significant changes in their quantity after IHNV infection, followed by sphingomyelins and glycolipids. Further analysis revealed that phosphatidylcholine (PC) was the lipid subclass that caused the most significant changes in its quantity after IHNV infection, followed by phosphatidylethanolamine (PE), phosphatidylserine (PS), and dimethylphosphatidylethanolamine (dMePE). In addition, IHNV infection caused significant changes in sphingomyelin (SM) and triglyceride (TG) subclasses. These results indicate that IHNV infection significantly alters the quantity and classification of lipid metabolites in host cells, with glycerolphospholipids showing the most significant changes.

[0061] 2. Differential changes in glycerophospholipid metabolites 72 hours after IHNV infection.

[0062] Further screening and analysis revealed that among 104 differentially expressed lipid metabolites induced by IHNV infection (VIP>1, fold change ≥1.2 or ≤0.83), 42 lipid metabolites were upregulated and 62 were downregulated. Figure 2 (A). These differentially metabolites are mostly glycerophospholipids, including PC, PE, SM, PS, and lysophosphatidylcholine (LPC). Results showed that IHNV infection significantly upregulated the molecular types of 36:4 (rep), 18:1 / 22:5, 22:5 / 22:6, 42:11, and 44:12 in cellular PC, and significantly downregulated the molecular types of 30:3 (rep), 31:3, 33:3, and 15:2 / 18:1 in PC, as well as the molecular types of 16:0, 30:0, and 36:0 in saturated long-chain PC (P<0.05). Viral infection also significantly upregulated the molecular types of 18:0 / 20:5, 17:1 / 22:6, 22:6, 18:2, and 20:2 / 22:6 in cellular PS (P<0.05). Furthermore, IHNV infection significantly downregulated the number of molecules in LPCs (14:0, 14:1, 16:0, 16:1, 17:1, 18:1, 16:2, 20:2, and 22:2), in LPIs (18:2, 20:2, 20:3, 20:4, 20:5, and 22:5), and in PEs (15:2 / 20:2, 15:2 / 18:1, 15:2 / 20:4, 15:2 / 18:1(rep), 15:2 / 20:4(rep), 15:2 / 22:6, 20:1 / 20:2, 36:1, 38:2e, and 40:2) (P<0.05). Figure 2 (B) For example Figure 2 The results showed that IHNV infection significantly reduced the total amount of lysophosphatidyl fatty acids such as LPG, LPC, LPI, LPS, and LPA in cells (P<0.05).

[0063] 3. Enrichment of differentially expressed lipid metabolites and screening of lipid-related differentially expressed genes (72 hours after IHNV infection)

[0064] 104 differentially expressed lipid metabolites were enriched into seven metabolic pathways, including glycerophospholipid metabolism, glycosylphosphatidylinositol (GPI)-anchored biosynthesis, glycerol metabolism, phosphatidylinositol signaling system, linoleic acid metabolism, α-linolenic acid metabolism, and arachidonic acid metabolism. Among these, glycerophospholipid metabolism was most significantly affected by IHNV (intra-Hyperactive Vulnerability). Figure 3 (A)

[0065] Based on RNA-seq transcriptomics data (PRJNA771177), the top 20 candidate genes with differential expression related to lipid metabolism were screened. Figure 3(B) Analysis showed that IHNV infection significantly upregulated the expression of choline transporter-like protein (CTL2, XM_021603363.1), ceramide synthase 1 (CerS1, XM_021559586.1), sphingomyelin synthase (SGMS2, ​​XM_021564774.1), phosphoethanolamine n-methyltransferase (MTCONS-00113722), and monoacylglycerol-O-acyltransferase (MOGAT2, XM_021592129.1) genes (P<0.05); and significantly inhibited the expression of neuraminidase 4 (NEU4, XM_021618682.1), β-galactosidase (GLB1, XM_021582848.1), and ceramide kinase ( The expression of genes such as CERK (XM_021563378.1), diacylglycerol kinase (DGKE, XM_021561048.1), phospholipase D2 (pLD2, XM_021573074.1), glycerol-3-phosphate-acyltransferase 3 / 4 (GPAT3, XM_021606457.1), lysophosphatidyltransferase 5 (LPCAT5, XM_021562060.1), SH3 domain-binding glutamate-like protein 3 (XM_021572255.1), and 1-acylglycerol-3-phosphate-acyltransferase (AGPAT, XM_021581392.1) was significantly reduced (P<0.05). These differentially expressed genes are mainly responsible for regulating glycerophospholipid metabolism. RT-qPCR analysis results were consistent with RNA-seq results. Figure 3 (C) verified the reliability of the sequencing data.

[0066] 4. The impact of AGPAT on IHNV replication

[0067] To investigate the effect of AGPAT on IHNV replication, we analyzed the expression levels of the AGPAT gene in cells 24 h, 36 h, 48 h, and 72 h after IHNV infection. The results showed that the expression level of the AGPAT gene was significantly higher than that of the MC group at 24 h and 36 h after IHNV infection, and significantly lower than that of the MC group at 48 h and 72 h after viral infection. Figure 4(P<0.05). The trend of AGPAT gene expression at 72 h after viral infection was consistent with the trend of AGPAT gene expression in the transcriptome data. We speculate that within 24 h and 36 h of viral infection, the virus mainly obtains a large amount of substances and energy to meet its own replication needs by promoting the expression of the AGPAT gene in the host cells and regulating the synthesis of phospholipids in the host cells. The downregulation of AGPAT gene expression after 48 h of viral infection is likely due to the fact that most cells have begun to disintegrate or even die, which leads to a decrease or loss of intracellular metabolic enzyme activity; or due to a decrease in the content of enzyme active substrates, resulting in a decrease in the activity of the corresponding enzymes.

[0068] Subsequently, we transfected RTG-2 cells with siRNA-AGPAT and infected them with IHNV for 24 h, 48 h, and 72 h, then examined the changes in IHNV-N gene expression and viral titer in host cells (i.e., the SiAGPAT-IH infection group). The experiment also included a silent virus infection negative control group (treated only with transfection reagent, i.e., the Si-IH infection group) and a virus infection negative control group (treated only with virus, i.e., the IH infection group). Figure 4 As shown in Table B, at 48h and 72h after IHNV infection, silencing the AGPAT gene significantly inhibited IHNV-N gene expression compared to the unsilenced AGPAT gene treatment group (P<0.05). This result indicates that AGPAT gene silencing can significantly inhibit IHNV replication. Furthermore, viral titer results also show that at 48h and 72h after IHNV infection, AGPAT gene silencing significantly reduced IHNV viral titers compared to the unsilenced AGPAT gene treatment group (Table 2) (P<0.05). This result also indicates that AGPAT gene silencing effectively inhibits IHNV replication. In conclusion, AGPAT can serve as a target for host resistance to IHNV for the prevention and treatment of IHNV infection.

[0069] Table 2. Effect of silencing the AGPAT gene on IHNV viral titer

[0070]

[0071] Note: Different letters indicate significant differences between groups (P<0.05).

[0072] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. Application of substances capable of inhibiting AGPAT gene expression in the preparation of products for treating infectious hematopoietic necrosis virus; The substance that can inhibit AGPAT gene expression is siRNA that targets the AGPAT gene. The siRNA targeting the AGPAT gene is a double-stranded RNA formed by SEQ ID No. 1 and SEQ ID No.

2.

2. The use of substances capable of inhibiting AGPAT gene expression in the preparation of products for the prevention and / or treatment of diseases caused by infectious hematopoietic necrosis virus infection; The substance that can inhibit AGPAT gene expression is siRNA that targets the AGPAT gene. The siRNA targeting the AGPAT gene is a double-stranded RNA formed by SEQ ID No. 1 and SEQ ID No.

2.

3. The application according to claim 2, characterized in that: The disease in question is infectious hematopoietic necrosis.

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