Application of phospholipase a2 as a target in prevention and treatment of infectious hematopoietic organ necrosis virus infection

Abstract

The application discloses application of phospholipase A2 as a target in prevention and treatment of infectious hematopoietic necrosis virus infection. The application provides application of a substance capable of inhibiting pLA2 gene expression in preparation of a product for resisting infectious hematopoietic necrosis virus. The application provides a new target and treatment scheme for preventing and treating occurrence and development of IHNV infection diseases, and has important significance for effectively improving the prevention and control level of rainbow trout virus diseases.

Description

Technical Field

[0001] This invention relates to the field of aquaculture, specifically to the application of phospholipase A2 as a target in the prevention and treatment of infectious hematopoietic necrosis virus infection. Background Technology

[0002] Infectious hematopoietic necrosis virus (IHNV) is a pathogen causing a highly fatal infectious disease in salmonid fish, resulting in a mortality rate exceeding 90% in juvenile rainbow trout and causing enormous economic losses to salmonid aquaculture worldwide. IHNV is a single-stranded, negative-sense RNA virus, an enveloped virus that encodes 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 is well known, viruses require the host's resources and capabilities to replicate and infect after invading host cells. As a typical enveloped virus, IHNV requires a large amount of lipids to provide sufficient energy and materials for its viral particle replication. Lipids are not only a source of energy in cells but also fundamental components of biological membranes. Phospholipids, including phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and phosphatidylinositol, are important components of the lipid bilayer and play a crucial role in viral replication and infection. Studies have reported that dengue virus, hepatitis B virus, poliovirus, grass carp reovirus, coronavirus, enterovirus, and influenza virus can reprogram host lipid metabolism processes by regulating enzymes involved in lipid metabolism to achieve their own replication and proliferation.

[0003] Our previous research found that IHNV infection significantly affects host cell glycerophospholipid metabolism, altering the types and quantities of phospholipid metabolites. Phospholipases are hydrolytic enzymes that break down phospholipids into various phosphatidic acids and amino alcohols, such as choline, serine, and ethanolamine. Based on the different sites of phospholipid hydrolysis, phospholipases are classified into phospholipases A1, A2, C, and D. Phospholipase A2 (pLA2) is a type of enzyme that specifically catalyzes the ester bond formed by the C2 hydroxyl group on the glycerol backbone of phospholipids (the acyl group at the sn-2 position of the glycerol molecule). It can hydrolyze a series of lipids, including PC, to produce free fatty acids, such as arachidonic acid. Arachidonic acid can then be oxidized by cyclooxygenases into a class of bioactive substances called arachidic acids, such as prostaglandins, leukotrienes, lysophosphatidic acid, and platelet-activating factor, which are lipid mediators. In vivo, these lipid mediators primarily regulate cell proliferation and migration, and also participate in physiological and pathological processes such as regulating intracellular and extracellular metabolism, maintaining membrane stability and permeability, and signal transduction by binding to G protein-coupled receptors. Therefore, pLA2, as the rate-limiting enzyme of these active substances, plays a crucial role in physiological and pathological processes such as immune responses and inflammatory reactions. However, the role of pLA2 in IHNV infection remains unclear, and the impact of the host cell pLA2 gene on viral replication after viral infection is also unknown. Therefore, clarifying the role of pLA2 in IHNV infection will help understand the influence of the virus on host cell phospholipid metabolism and provide new targets for the prevention and treatment of IHNV infection. Summary of the Invention

[0004] The purpose of this invention is to provide the application of phospholipase A2 (pLA2) as a target in the prevention and treatment of infectious hematopoietic organ necrosis virus (IHNV) infection.

[0005] In a first aspect, the present invention claims the use of a substance capable of inhibiting pLA2 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 pLA2 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] The disease in question is infectious hematopoietic necrosis.

[0008] Thirdly, the present invention claims protection for the use of a substance capable of inhibiting pLA2 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 pLA2 gene expression in the preparation of cell or animal models with enhanced resistance to infectious hematopoietic organ necrosis virus (IHNV).

[0013] In the above aspects, the substance that can inhibit pLA2 gene expression is a substance that directly targets the pLA2 gene and can reduce pLA2 gene expression.

[0014] Furthermore, the substance capable of inhibiting pLA2 gene expression may be siRNA targeting the pLA2 gene.

[0015] Furthermore, the siRNA targeting the pLA2 gene is a double-stranded RNA formed by SEQ ID No. 1 and SEQ ID No. 2.

[0016] 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).

[0017] The method for preparing a cell model or animal model with enhanced resistance to infectious hematopoietic organ necrosis virus (IHNV) claimed in this invention includes the following steps: reducing the expression of the pLA2 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.

[0018] Specifically, reducing the expression of the pLA2 gene in the host cells or animals can be achieved by introducing siRNA targeting the pLA2 gene into the host cells or animals.

[0019] Furthermore, the siRNA targeting the pLA2 gene may be a double-stranded RNA formed by SEQ ID No. 1 and SEQ ID No. 2.

[0020] Sixthly, the present invention claims protection for any of the following applications:

[0021] (B1) Application of pLA2 gene as a target in regulating the resistance of host cells or animals to infectious hematopoietic necrosis virus (IHNV);

[0022] (B2) Application of pLA2 gene as a target in regulating the replication ability of infectious hematopoietic necrosis virus (IHNV) in host cells or animals;

[0023] (B3) Application of pLA2 gene as a target in the preparation of products for regulating the resistance of host cells or animals to infectious hematopoietic necrosis virus (IHNV);

[0024] (B4) Application of the pLA2 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.

[0025] In (B1) and (B2), the application may be a non-disease diagnosis or treatment application.

[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 pLA2 gene is shown in SEQ ID No. 3.

[0031] This invention provides new targets and treatment options 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 This represents the change in pLA2 gene expression during IHNV infection.

[0033] Figure 2 The changes in PC content in IHNV infection.

[0034] Figure 3 To investigate the effect of silencing the pLA2 gene on the expression level of the IHNV-N gene.

[0035] Figure 4 The effect of the pLA2 gene on the PC content in cells.

[0036] In each figure, * indicates a significant difference compared to the corresponding control group (P<0.05). Detailed Implementation

[0037] 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.

[0038] 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.

[0039] Example 1: Application of phospholipase A2 as a target in the prevention and treatment of infectious hematopoietic organ necrosis virus infection 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), 15°C, 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 strain (MOI 1.0) and incubated at 15°C with 5% CO2 to establish an IHNV challenge model. Uninfected cells served as a control. Cells from the IHNV challenge group (IH group) and the control group (MC group) were collected at 24h, 36h, 48h, and 72h post-infection.

[0045] 3. PC content determination

[0046] With a density of 1×10 5RTG-2 cells were seeded in 6-well plates and cultured overnight in complete medium. An IHNV challenge model (MOI 1.0) was established using the IHNV-Sn1203 strain, with a control group (i.e., cells not infected with IHNV) included. Cells from the IHNV challenge group (IH group) and the control group (MC group) were collected at 24h, 36h, 48h, and 72h after IHNV infection for PC content assay (Phosphatidylcholine Assay Kit, abcam).

[0047] 4. RT-qPCR analysis

[0048] 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. Primers are shown in Table 1. Using β-actin as an internal control, the relative expression levels of gene transcripts (2T) were calculated using the comparative cycle threshold (Ct) method. -ΔΔCt ).

[0049] Table 1. RT-qPCR primers

[0050]

[0051] 5. pLA2 RNA interference (RNAi)

[0052] The nucleotide sequence of the pLA2 gene is shown in SEQ ID No. 3.

[0053] Specific siRNA oligonucleotides targeting pLA2 (5'-GCACCUUUCAUCCGGAAAUTT-3' (SEQ ID No. 1), 5'-AUUUCCGGAUGAAAGGUGCTT-3' (SEQ ID No. 2)) 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 interference reagent were added to Opti-MEM medium and mixed thoroughly at room temperature for 5 min. Subsequently, the siRNA-pLA2 mixture 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. A silencing negative control group (only transfection reagent was added, and the infected group was the si-IH infection group) and a negative control group (only virus treatment group, i.e., the IH infection group) were also included. Viral titers were measured using TCID50. 50 The method was used for determination.

[0054] II. Results and Analysis

[0055] 1. Changes in pLA2 in IHNV infection

[0056] To investigate the changing trend of pLA2 in IHNV infection, we analyzed the expression level of the pLA2 gene in cells (i.e., the IH group) 24h, 36h, 48h, and 72h after IHNV infection. The results showed that compared with the control (MC group), the expression level of the pLA2 gene in the IH group cells was significantly increased at 24h, 36h, and 48h after IHNV infection, and significantly decreased at 72h after viral infection. Figure 1 (P<0.05). For example... Figure 2 As shown, compared with the MC group, the PC content in IH group cells was significantly reduced at 24h and 36h after IHNV infection (P<0.05). pLA2 is a specific hydrolase that catalyzes a series of phospholipids, including PC. Therefore, it is speculated that the virus may regulate intracellular PC hydrolysis by promoting the expression of the pLA2 gene in host cells, thus leading to a decrease in PC content. The PC content showed an increasing trend after 48h of viral infection, which is likely due to the fact that most cells had begun to disintegrate or even die, leading to a decrease or loss of intracellular metabolic enzyme activity. The continuous reduction in the content of enzyme active substrates leads to a decrease in the corresponding enzyme activity.

[0057] 2. Effects of pLA2 on IHNV-N gene expression

[0058] RTG-2 cells were transfected with siRNA-pLA2 and infected with IHNV for 24, 36, 48, and 72 hours. The expression of the IHNV-N gene in host cells was then measured (i.e., the SipLA2-IH group). Analysis of the relative expression level of the pLA2 gene revealed that, compared with the silencing control group (treatment only with the transfection reagent), siRNA-pLA2 significantly reduced pLA2 gene expression (a decrease of 58%), confirming the successful construction of a pLA2 gene silencing model. Figure 3 As can be seen, at 24h, 36h, 48h, and 72h after IHNV infection, compared with the control group (i.e., the Si-IH group), the silencing of the pLA2 gene (i.e., the SipLA2-IH group) significantly inhibited the expression of the IHNV-N gene in host cells. This result indicates that the silencing of the pLA2 gene significantly inhibits the replication of IHNV (P<0.05).

[0059] 3. The effect of pLA2 on viral titer

[0060] RTG-2 cells were transfected with siRNA-pLA2 and infected with IHNV at 24h, 36h, 48h, and 72h, and viral titers were measured (i.e., the SipLA2-IH group). The viral titer results also showed that silencing the pLA2 gene at 48h and 72h after IHNV infection significantly reduced the IHNV viral titer (Table 2) (P<0.05). This result indicates that pLA2 silencing effectively inhibits IHNV infection. In conclusion, pLA2 can serve as a target for host resistance to IHNV for the prevention and treatment of IHNV infection.

[0061] Table 2. Effect of silencing the pLA2 gene on IHNV viral titer

[0062]

[0063] Note: Different lowercase letters marked on different groups within the same viral infection time period indicate significant differences between them (P<0.05).

[0064] 4. Effect of pLA2 on PC content in cells

[0065] RTG-2 cells were transfected with siRNA-pLA2 and infected with IHNV for 24 h, 36 h, 48 h, and 72 h, at which time the PC content in host cells was measured. Figure 4It can be seen that the PC content in the SipLA2-IH group cells was significantly higher than that in the IH and Si-IH groups at 24h, 36h, and 48h after IHNV infection, with significant differences at 24h and 48h (P<0.05). At 72h after infection, the PC content did not change significantly in the three treatment groups. This result indicates that silencing the pLA2 gene inhibits the hydrolysis of PC in IHNV-infected host cells. These results suggest that during viral infection, IHNV can regulate pLA2 gene expression to hydrolyze PC, thereby generating the raw materials and energy required for viral particle replication.

[0066] 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. The application of substances capable of inhibiting pLA2 gene expression in any of the following: (A1) Prepare a product for inhibiting the replication of infectious hematopoietic necrosis virus in host cells; (A2) Prepare a product for reducing the titer of infectious hematopoietic necrosis virus at the cellular level; The substance that can inhibit pLA2 gene expression is a substance that directly targets the pLA2 gene and can reduce pLA2 gene expression; The substance that can inhibit pLA2 gene expression is siRNA that targets the pLA2 gene; The siRNA targeting the pLA2 gene is a double-stranded RNA formed by SEQ ID No. 1 and SEQ ID No. 2; The cells are fish cells; The fish cells in question are RTG-2 cells.

2. Application of substances that can inhibit pLA2 gene expression in the preparation of cell models with enhanced resistance to infectious hematopoietic necrosis virus; The substance that can inhibit pLA2 gene expression is a substance that directly targets the pLA2 gene and can reduce pLA2 gene expression; The substance that can inhibit pLA2 gene expression is siRNA that targets the pLA2 gene; The siRNA targeting the pLA2 gene is a double-stranded RNA formed by SEQ ID No. 1 and SEQ ID No. 2; The cells are fish cells; The fish cells in question are RTG-2 cells.

3. The application according to claim 1 or 2, characterized in that: The nucleotide sequence of the pLA2 gene is shown in SEQ ID No.

3.

4. A method for preparing a cell model with enhanced resistance to infectious hematopoietic necrosis virus, comprising the following steps: reducing the expression of the pLA2 gene in host cells to obtain recombinant cells; and the recombinant cells exhibiting enhanced resistance to infectious hematopoietic necrosis virus compared to the host cells. The reduction of pLA2 gene expression in the host cells is achieved by introducing siRNA targeting the pLA2 gene into the host cells; The siRNA targeting the pLA2 gene is a double-stranded RNA formed by SEQ ID No. 1 and SEQ ID No. 2; The cells are fish cells; the fish cells are RTG-2 cells.

5. The method according to claim 4, characterized in that: The nucleotide sequence of the pLA2 gene is shown in SEQ ID No.

3.

6. A cell model prepared using the method described in claim 4 or 5.

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