Methods for promoting viral replication of cheetah herpesvirus through signaling pathway-mediated innate immune escape

Abstract

This invention discloses a method for promoting viral replication of CyHV-3 through innate immune escape mediated by a signaling pathway, comprising the following steps: First, preparation of materials is required, including cells and virus: Common carp brain (CCB) cells are cultured in M199 medium supplemented with 10% fetal bovine serum (FBS, Life Technologies) at 28°C under a 5% CO2 atmosphere, while human embryonic kidney 293T cells are cultured in DMEM medium containing 10% FBS. This invention identifies ORF24 as a key immune escape factor for CyHV-3 virus. We demonstrate that ORF24 can significantly inhibit IFN expression and promote viral replication. Mechanistically, ORF24 directly interacts with multiple components of the RLR pathway (including MITA, TBK1, IRF3, and IRF7) without affecting the stability of these proteins. Instead, ORF24 inhibits the recruitment of IRF3 / 7 by MITA and TBK1, thereby blocking RLR-mediated IFN signaling. Our study reveals that CyHV-3 ORF24 reveals a novel mechanism by which it circumvents the host's innate immunity and provides a potential molecular target for developing antiviral strategies against CyHV-3 infection.

Description

Technical Field

[0001] This invention relates to the field of carp herpesvirus research technology, specifically to a method for promoting viral replication of carp herpesvirus through signaling pathway-mediated innate immune escape. Background Technology

[0002] As a member of the Herpesviridae family, CyHV-3, also known as Koi herpesvirus (KHV), is a highly contagious pathogen that can infect common carp, koi and their hybrid varieties. The virus spreads rapidly and has extremely high morbidity and mortality rates, causing huge economic losses to the global carp aquaculture industry. Therefore, it has become a key target for disease prevention and control in the field of aquatic virology [1]. The virus was first discovered in Germany in the late 1990s and caused a large-scale death of koi. Since then, it has spread globally [2]. The virus has a 295 kb genome that encodes 164 open reading frames (ORFs), which is the largest known genome of all herpesviruses [3].

[0003] After viral infection, host pattern recognition receptors (PRRs) can recognize viral nucleic acids and proteins, thereby initiating an innate immune response and inducing the production of interferon (IFN) [4,5]. In fish, the evolutionarily conserved RIG-I-like receptor (RLR) pathway triggers type I IFN responses through IRF3 / 7-dependent signal transduction [6,7]. RLRs (such as RIG-I and MDA5) can recognize viral RNA and activate the adaptor protein MAVS, which then recruits MITA, TRAF3, and TBK1. These proteins phosphorylate IRF3 / 7, and these transcription factors are then transported to the cell nucleus, where they bind to interferon-stimulated response elements (ISREs) and drive the expression of type I interferon and IFN-stimulated genes (ISGs), thereby establishing an antiviral state [8-10]. In contrast, viruses have evolved a variety of strategies to evade host immunity, and the balance between host defense and viral immune escape determines the infection outcome

[11] .

[0004] Herpesviruses belong to the double-stranded DNA virus family and are highly infectious, posing a serious threat to human and animal health

[12] . During their continuous evolution, these viruses have also developed a variety of immune evasion strategies. In humans, human gamma herpesviruses are usually carcinogenic and can induce cancers with poor prognosis[13,14]. UL36 is a protein encoded by HCMV that can inhibit the phosphorylation and activation of IRF3, thereby antagonizing the IRF3-mediated innate immune response and blocking antiviral signal transduction

[15] . In animals, the selective and precise autophagy regulation mechanism mediated by the kinase-like protein (KLP) encoded by Nautilus herpesvirus type 2 (NaHV-2) limits the production of interferon (IFN) by degrading MITA

[16] . The envelope protein (E) of porcine acute diarrhea syndrome coronavirus (SADS-CoV) induces STAT2 degradation through macroautophagy / autolysosome pathway, thereby inhibiting type I interferon (IFN-I) signal transduction

[17] .Similar immune escape phenomena have also been reported in fish: the extracellular domain of the ORF4 protein encoded by carp herpesvirus type 2 (CyHV-2) is highly homologous to the host tumor necrosis factor receptor (TNFRs). This protein can “mimic” the receptor molecule and competitively bind to the key pro-inflammatory cytokine TNF-α secreted by the host, blocking its normal binding to endogenous TNFRs

[18] , thereby inhibiting IFN. The VP6 protein of grass carp reovirus (GCRV) degrades TANK-binding kinase 1 (TBK1) via autophagy, thereby implementing an immune escape strategy to inhibit the activation of interferon (IFN)

[19] . For cyprinid herpesvirus type 3 (CyHV-3), viral DNA is most often detected in brain tissue samples, and no evidence of viral replication has been found, suggesting that the brain may be a latent infection site for CyHV-3

[21] . Carp is one of the most economically valuable farmed fish in China

[22] , but the outbreak of CyHV-3 virus has seriously hindered the further development of aquaculture

[23] . Currently, a rapid viral replication method based on nanotechnology has been established

[24] . The viral interleukin-10 (v) encoded by the CyHV-3 virus ORF134 gene is used to generate and weaken the host's early antiviral immune response. IL-10 protein can inhibit LPS-induced pro-inflammatory gene expression, thereby promoting viral immune escape

[25] . CyHV-3's ORF4 and ORF12 proteins are homologs of viral tumor necrosis factor receptors (vTNFRs), which can induce apoptosis during host

[20] viral infection, thereby achieving immune escape

[26] . In addition, recent studies have shown that CyHV-3's ORF24 protein has no significant sequence similarity to other cyprinid herpesvirus homologs, suggesting that this gene may have evolved a unique functional role during viral evolution. Existing studies have confirmed that ORF24 can effectively inhibit the expression of host immune factors, but the exact molecular mechanism of its action has not yet been elucidated

[27] . Therefore, in-depth research on the immune escape strategy adopted by CyHV-3 is of great significance for the prevention and control of related viral diseases. Summary of the Invention

[0005] The purpose of this invention is to provide a method for promoting viral replication of cheetah herpesvirus through signaling pathway-mediated innate immune escape, in order to solve the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a method for promoting viral replication of cheetah herpesvirus through signaling pathway-mediated innate immune escape, the replication method comprising the following steps: S1. First, the materials need to be prepared, including cells and viruses: Common carp brain (CCB) cells are cultured in M199 medium supplemented with 10% fetal bovine serum (FBS, Life Technologies) at 28°C and 5% CO2 atmosphere; human embryonic kidney 293T cells are cultured in DMEM medium (Life Technologies) containing 10% FBS at 37°C and 5% CO2 atmosphere. For virus amplification, CCB cells are infected with CyHV-3 virus, and the collected virus samples are stored at -80°C. Reagents and antibodies are also included: negative control (NC), the sequences of NC are 5′-uucuccgaacgugucactt-3′ (forward) and 5′-acgugacacguucggagaat-3′ (reverse); mouse anti-Flag, anti-hemagglutinin (HA), anti-Myc and anti-enhanced green fluorescent protein (EGFP) antibodies.

[0007] S2. Next, plasmid construction is required: Total RNA was extracted from CyHV-3-infected CCB cells using TRIzol reagent (Invitrogen). The open reading frame (ORF) of ORF24 (GenBank ID: 11266358) was amplified by reverse transcription polymerase chain reaction (RT-PCR) and cloned into the pEGFP-N1 vector. All plasmids were constructed according to the specified method, and all constructs were verified by DNA sequencing. Luciferase activity assay is required: CCB cells were seeded in 24-well plates and cultured for 12 hours. Subsequently, plasmids expressing MAVS, TBK1, MITA, IRF3, or IRF7 were combined with IFNφ1pro-Luc, IFNφ2pro-Luc, or IFNφ3pro-Luc. - Luc or ISREpro-Luc reporter plasmid (including pRL-TK Renilla luciferase plasmid as an internal control) were co-transfected into cells. 24 hours after transfection, cells were washed with phosphate-buffered saline (PBS), and luciferase activity was measured using the Dual-Luciferase Reporter Assay Kit (YEASEN) according to the manufacturer's instructions. To ensure the accuracy of subsequent data analysis, firefly luciferase activity was standardized to Renilla luciferase activity.

[0008] S3. Next, transfection is required. Cells in the 12-well plate are transfected using Trans-Introduction. Transfection was performed using EL transfection reagent (TransGen Biotech) in 200 μL of optimized serum-free medium. Six hours post-transfection, the medium was replaced with 1 mL of M199 medium or Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Cells were then cultured for another 18 hours before viral infection experiments: CCB cells were inoculated with CyHV-3 virus at a multiplicity of infection (MOI) of 10. Control cells were simultaneously treated with an equal volume of phosphate-buffered saline (PBS). All cells were then cultured according to the same protocol, and relevant parameters were measured. After adsorption at 25°C for 2 hours, the inoculum was removed, and the medium was replaced with M199 medium containing 5% fetal bovine serum (FBS). Cells were collected at 12, 24, 36, and 48 hours post-infection, and viral mRNA levels were detected using quantitative real-time polymerase chain reaction (qRT-PCR).

[0009] S4. Next, RNA extraction, reverse transcription, and qRT-PCR are required. Total RNA is extracted using TRIzol reagent. PrimeScript is used according to the manufacturer's instructions. First-strand complementary DNA (cDNA) was synthesized using an RT kit (containing gDNA Eraser, Takara Bio). Quantitative real-time PCR (qRT-PCR) was performed on a CFX96 real-time quantitative PCR system (Bio-Rad) using AceQ. qPCR SYBR Green premix (Vazyme Biotech) is used for Western blot analysis: Total cellular proteins are extracted using cell lysis buffer (containing 50 mM Tris [pH 7.4], 150 mM sodium chloride, 1% Triton X-100, 1% sodium deoxycholate, and 0.1% SDS). Protein samples are denatured at 100°C for 10 minutes, separated by 10% SDS-PAGE gel electrophoresis, and then transferred to a nitrocellulose membrane (Biosharp). The membrane is blocked overnight at 4°C in Tris-buffered saline (TBST) containing 5% skim milk powder, followed by incubation with the specified primary antibody (Abs) at room temperature for 2 hours. After washing three times with TBST, the membrane is then incubated with IRDye at room temperature. Incubate with 800CW-labeled donkey anti-rabbit IgG (1:10,000) or anti-mouse IgG (1:10,000) for 1 hour, and detect signal intensity using an Amersham Imager 680 (Cytiva).

[0010] S5. The co-immunoprecipitation experiment needs to be performed in sequence: Seed an appropriate amount of cells into a culture flask and culture until the cell density reaches 70-80% confluence. Then, transfect the cells with a total of 16 μg of target plasmid. After culturing for 24 hours, add 2 mL of ice-cold phosphate-buffered saline (PBS), gently wash the cell monolayer twice, scrape the cells off the culture flask wall with a cell scraper, collect the cell suspension and transfer it to a centrifuge tube. Centrifuge at 1000 rpm for 5 minutes and collect the cell pellet. Add pre-chilled PBS to the pellet, gently resuspend the cells using a pipette, and centrifuge again at 1000 rpm for 3 minutes. Discard the supernatant to obtain a purified cell pellet. Add an equal volume of lysis buffer (Sigma-Aldrich, St. Louis, Missouri, USA) to the pellet volume, mix the cells thoroughly by repeated pipetting, and incubate on ice for 10–20 minutes to achieve complete lysis. Transfer the lysis buffer to a centrifuge tube and centrifuge at 12,000 × g for 15 minutes at 4°C. After centrifugation, collect the supernatant containing soluble protein, discard the cell debris at the bottom, and transfer the collected protein supernatant to a new centrifuge tube. Add 30 μL of anti-Flag affinity gel (DIA-AN), place the centrifuge tube in a shaker, and gently vortex overnight at 4°C to allow the Flag-labeled protein to fully bind to the affinity gel. After incubation, collect the affinity gel bound to the target protein by centrifuging at 5000 × g for 3 minutes at 4°C and discard the supernatant. Add an appropriate amount of ice-cold PBS to the gel, gently resuspend, centrifuge again, discard the supernatant, and repeat this washing step three times to remove non-specifically bound contaminating proteins. Add 25 μL of 5×SDS sample buffer to the washed affinity gel, resuspend and mix thoroughly to obtain immunoprecipitated protein samples. After subsequent processing, perform Western blot (IB) analysis on the samples to further detect proteins.

[0011] S6. Finally, confocal imaging is required: 293T cells are seeded on glass coverslips and transfected with plasmids for 24 hours. The cells are then washed twice with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde for 20 minutes, washed three more times with PBS, and stained with 1 mg / mL 4′,6-diamidinyl-2-phenylindole (DAPI, Beyotime) for 15 minutes at room temperature in the dark. Finally, the cells were imaged using a Nikon laser scanning confocal microscope (N-storm; Nikon super-resolution microscope). Imaging was performed using an electron multiplication charge-coupled device (EMCCD) camera (iXonDU-897, Andor) and a 100×, numerical aperture (NA) TIRF objective lens (CFI Apochromat TIRF, Nikon). Fluorescence signals were excited by a 488 nm laser and collected through a bandpass emission filter (500–545 nm). Image acquisition and reconstruction were performed using Nikon NIS-Elements software, and the experimental data were statistically analyzed.

[0012] Preferably, the plasmids pCDNA-MAVS (NM_001080584.2), pCMV-Tag2C-MAVS, pCDNA-TBK1 (NM_001044748.2), pCMV-Tag2C-TBK1, pDsRed-TBK1, pCDNA-MITA (NM_001278837.1), pCMV-Tag2C-MITA, pDsRed-MITA, pCDNA-IRF3 (NM_001143904), and p CMV-Tag2C-IRF3, pDsRed-IRF3, pCDNA-IRF7 (NM_200677.2), pCMV-Tag2C-IRF7, pDsRed-IRF7, as well as IFNφ1pro-Luc, IFNφ2pro-Luc, IFNφ3pro-Luc, interferon-stimulated response element (ISRE)-Luc, pMyc-IRF3, and pHA-IRF7 were constructed according to the previously described method, and all constructs were validated by DNA sequencing.

[0013] Preferably, the formula for calculating the standardized luciferase activity is as follows: Standardized luciferase activity = Firefly luciferase reading ÷ Corresponding Renida luciferase reading.

[0014] Preferably, the PCR cycling conditions are as follows: initial denaturation at 95°C for 5 minutes; followed by 40 cycles, each cycle consisting of denaturation at 95°C for 10 seconds, annealing at 60°C for 10 seconds, and extension at 72°C for 15 seconds. After amplification, melting curve analysis is performed at a heating rate of 5°C / s from 95°C to verify the specificity of the PCR product. - The ΔΔCt method (double ΔCt method) normalizes the data of each sample to the level of β-actin (internal reference gene).

[0015] Preferably, all statistical analyses were performed using GraphPad Prism 5.0 (GraphPad Software). Statistical significance was determined by a two-tailed unpaired Student t-test, with p < 0.05 considered statistically significant. All data represented the results of at least two independent experiments, with each experiment repeated three times (mean ± standard deviation). An asterisk (*) indicated a statistically significant difference (*p < 0.05, **p < 0.01).

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention identifies ORF24 as a key immune escape factor for CyHV-3 virus. We demonstrate that ORF24 can significantly inhibit IFN expression and promote viral replication. Mechanistically, ORF24 directly interacts with multiple components of the RLR pathway (including MITA, TBK1, IRF3, and IRF7) without affecting the stability of these proteins. Instead, ORF24 hinders the recruitment of IRF3 / 7 by MITA and TBK1, thereby blocking RLR-mediated IFN signaling. Our study reveals a novel mechanism by which CyHV-3 ORF24 evades the host's innate immunity and provides a potential molecular target for developing antiviral strategies against CyHV-3 infection.

[0017] This invention confirms that the early protein ORF24 of CyHV-3 virus plays a key role in virus-host interaction and immune escape. Experiments show that ORF24 promotes viral immune escape by weakening the ability of MITA and TBK1 to recruit IRF3 / 7 and inhibiting the production of host IFN. The discovery of the ORF24 immune escape mechanism lays the foundation for the future development of anti-CyHV-3 drugs and disease detection methods. Attached Figure Description

[0018] Figure 1 This is a statistical table showing the effect of viral immediate early protein overexpression on IFN promoter activity and viral replication according to the present invention. Figure 2The ORF24 of this invention promotes CyHV-3 replication and inhibits cellular IFN expression statistics. Figure 3 This is a statistical table showing how ORF24 can inhibit the expression of interferons (IFNs) and interferon-stimulated genes (ISGs) according to the present invention; Figure 4 The ORF24 of this invention blocks the activation statistics of the RLR-mediated IFN signaling pathway; Figure 5 This is a schematic diagram illustrating the interaction between ORF24 and STM molecules according to the present invention; Figure 6 This is a schematic diagram of the MITA and TBK1 recruitable IRF3 / 7 in Cyprinus carpio of the present invention. Figure 7 This is a schematic diagram illustrating how overexpression of ORF24 in this invention impairs the recruitment effect of MITA and TBK1 on IRF3 / 7 in a dose-dependent manner. Detailed Implementation

[0019] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] Please see Figure 1-7 The herpesvirus cypriniform promotes viral replication through signaling pathway-mediated innate immune escape. This replication method includes the following steps: S1. First, the materials need to be prepared, including cells and viruses: Common carp brain (CCB) cells were cultured in M199 medium supplemented with 10% fetal bovine serum (FBS, Life Technologies) at 28°C and 5% CO2. Human embryonic kidney 293T cells were cultured in DMEM medium (Life Technologies) containing 10% FBS at 37°C and 5% CO2. For virus amplification, CCB cells were infected with CyHV-3 virus, and the collected virus samples were stored at -80°C. Reagents and antibodies were also included: negative control (NC), with sequences of 5′-uucuccgaacgugucactt-3′ (forward) and 5′-acgugacacguucggagaat-3′ (reverse). Mouse anti-Flag, anti-hemagglutinin (HA), anti-Myc, and anti-enhanced green fluorescent protein (EGFP) antibodies were also prepared. This study constructed a protein encoding the immediate early stage of CyHV-3 (including EGFP-ORF1, EGFP-ORF1, and EGFP-ORF2). Eukaryotic expression plasmids (EGFP-ORF3, EGFP-ORF6, EGFP-ORF7, EGFP-ORF11, EGFP-ORF24, EGFP-ORF54, EGFP-ORF88, EGFP-ORF146, and EGFP-ORF149) were developed. These plasmids, along with polyinosinic-cytosine monophosphate (poly(I:C)) and an IFN promoter reporter plasmid, were co-transfected into cells. The effects of these viral proteins on IFN promoter activity were assessed using a dual-luciferase reporter gene assay. Results showed that after poly(I:C) stimulation, IFN promoter activity was significantly reduced in cells overexpressing ORF1, ORF7, ORF24, or ORF146 compared to the empty vector control group. Meanwhile, after transfecting each viral protein expression plasmid into CCB cells, the cells were infected with CyHV-3 for 48 hours. Subsequently, the mRNA expression level of the viral gene ORF12 was detected. The results showed that overexpression of ORF24 had the most significant promoting effect on CyHV-3 replication. In summary, these findings suggest that ORF24 may promote CyHV-3 proliferation by negatively regulating the IFN response.

[0021] S2. Next, plasmid construction is required: Total RNA was extracted from CyHV-3-infected CCB cells using TRIzol reagent (Invitrogen). The open reading frame (ORF) of ORF24 (GenBank ID: 11266358) was amplified by reverse transcription polymerase chain reaction (RT-PCR) and cloned into the pEGFP-N1 vector. All plasmids were constructed according to the specified method, and all constructs were verified by DNA sequencing. Luciferase activity assay is required: CCB cells were seeded in 24-well plates and cultured for 12 hours. Subsequently, plasmids expressing MAVS, TBK1, MITA, IRF3, or IRF7 were combined with IFNφ1pro-Luc, IFNφ2pro-Luc, or IFNφ3pro-Luc. -Luc or ISREpro-Luc reporter plasmids (including the pRL-TK Renilla luciferase plasmid as an internal control) were co-transfected into cells. Twenty-four hours after transfection, cells were washed with phosphate-buffered saline (PBS), and luciferase activity was measured using the Dual-Luciferase Reporter Assay Kit (YEASEN) according to the manufacturer's instructions. To ensure the accuracy of subsequent data analysis, firefly luciferase activity was normalized to Renilla luciferase activity. CCB cells were transfected with a eukaryotic expression plasmid encoding ORF24. Twenty-four hours after transfection, cells were infected with CyHV-3 virus, and cells were collected 48 hours post-infection to detect viral gene and IFN expression levels. Compared with the control group, overexpression of ORF24 significantly upregulated the mRNA levels of viral ORF4, ORF7, ORF12, and MCP. Figure 2 A) indicates that ORF24 can effectively promote CyHV-3 proliferation; at the same time, the mRNA expression level of IFN is significantly reduced ( Figure 2 B) indicates that ORF24 significantly inhibits IFN expression during CyHV-3 infection.

[0022] S3. Next, transfection is required. Cells in the 12-well plate are transfected using Trans-Introduction. Transfection was performed using EL transfection reagent (TransGen Biotech) in 200 μL of optimized serum-free medium. Six hours after transfection, the transfection medium was replaced with 1 mL of M199 or Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Cells were then cultured for another 18 hours before viral infection experiments were performed: CCB cells were inoculated with CyHV-3 virus at a multiplicity of infection (MOI) of 10. Control group cells were treated with an equal volume of phosphate-buffered saline (PBS). All cells were then cultured and relevant indicators were measured according to the same protocol. After adsorption at 25°C for 2 hours, the inoculum was removed and replaced with M199 medium containing 5% fetal bovine serum (FBS). Cells were collected at 12, 24, 36, and 48 hours post-infection, and viral mRNA levels were detected using quantitative real-time polymerase chain reaction (qRT-PCR). To investigate whether ORF24 regulates IFN expression through the classical RLR signaling pathway, researchers examined the effect of ORF24 on the expression of IFN and its downstream effector ISRE. Figure 4 As shown, the expression levels of IFN and ISRE increased after poly(I:C) stimulation, indicating that downstream RLR signaling molecules can activate the promoter activity of IFN and ISRE in CCB cells. Conversely, transfection with ORF24 significantly reduced the expression of IFN and ISRE, suggesting that ORF24 not only inhibits IFN expression but also downregulates the expression of downstream antiviral genes, thereby weakening the host's antiviral immune response. These results further suggest that ORF24 may affect IFN expression by regulating components of the RLR signaling pathway.

[0023] S4. Next, RNA extraction, reverse transcription, and qRT-PCR are required. Total RNA is extracted using TRIzol reagent. PrimeScript is used according to the manufacturer's instructions. First-strand complementary DNA (cDNA) was synthesized using an RT kit (containing gDNA Eraser, Takara Bio). Quantitative real-time PCR (qRT-PCR) was performed on a CFX96 real-time quantitative PCR system (Bio-Rad) using AceQ. qPCR SYBR Green premix (Vazyme Biotech) is used for Western blot analysis: Total cellular proteins are extracted using cell lysis buffer (containing 50 mM Tris [pH 7.4], 150 mM sodium chloride, 1% Triton X-100, 1% sodium deoxycholate, and 0.1% SDS). Protein samples are denatured at 100°C for 10 minutes, separated by 10% SDS-PAGE gel electrophoresis, and then transferred to a nitrocellulose membrane (Biosharp). The membrane is blocked overnight at 4°C in Tris-buffered saline (TBST) containing 5% skim milk powder, followed by incubation with the specified primary antibody (Abs) at room temperature for 2 hours. After washing three times with TBST, the membrane is then incubated with IRDye at room temperature. Incubation with 800CW-labeled donkey anti-rabbit IgG (1:10,000) or anti-mouse IgG (1:10,000) for 1 hour followed by signal intensity detection using an Amersham Imager 680 (Cytiva) was conducted. To further clarify the role of ORF24 in the IFN signaling pathway, researchers utilized the key RLR cascade reaction in the IFN activation process to investigate whether and how ORF24 regulates RLR-mediated IFN signaling in cells. Figure 5 As shown, overexpression of MAVS, MITA, TBK1, IRF3, and IRF7 significantly enhanced the promoter activities of IFN1φpro, IFNφ3pro, and ISRE; however, overexpression of ORF24 effectively inhibited this activation effect, indicating that ORF24 has a negative regulatory effect on IFN expression activated by RLR signaling molecules.

[0024] S5. The co-immunoprecipitation experiment needs to be performed in sequence: Seed an appropriate amount of cells into a culture flask and culture until the cell density reaches 70-80% confluence. Then, transfect the cells with a total of 16 μg of target plasmid. After culturing for 24 hours, add 2 mL of ice-cold phosphate-buffered saline (PBS), gently wash the cell monolayer twice, scrape the cells off the culture flask wall with a cell scraper, collect the cell suspension and transfer it to a centrifuge tube. Centrifuge at 1000 rpm for 5 minutes and collect the cell pellet. Add pre-chilled PBS to the pellet, gently resuspend the cells using a pipette, and centrifuge again at 1000 rpm for 3 minutes. Discard the supernatant to obtain a purified cell pellet. Add an equal volume of lysis buffer (Sigma-Aldrich, St. Louis, Missouri, USA) to the pellet volume, mix the cells thoroughly by repeated pipetting, and incubate on ice for 10–20 minutes to achieve complete lysis. Transfer the lysis buffer to a centrifuge tube and centrifuge at 12,000 × g for 15 minutes at 4°C. After centrifugation, collect the supernatant containing soluble protein, discard the cell debris at the bottom, and transfer the collected protein supernatant to a new centrifuge tube. Add 30 μL of anti-Flag affinity gel (DIA-AN), place the centrifuge tube in a shaker, and gently vortex overnight at 4°C to allow the Flag-labeled protein to fully bind to the affinity gel. After incubation, collect the affinity gel bound to the target protein by centrifuging at 5000 × g for 3 minutes at 4°C and discard the supernatant. Add an appropriate amount of ice-cold PBS to the gel, gently resuspend, centrifuge again, discard the supernatant, and repeat this washing step three times to remove non-specifically bound contaminating proteins. Add 25 μL of 5×SDS sample buffer to the washed affinity gel, resuspend thoroughly, and mix to obtain immunoprecipitated protein samples. After subsequent processing, the samples are analyzed by Western blotting (IB) to further detect proteins. To further verify the interaction between ORF24 and STM molecules, ORF24-EGFP was co-transfected with Flag-MAVS, Flag-MITA, Flag-TBK1, Flag-IRF3, or Flag-IRF7 into 293T cells, and co-immunoprecipitation experiments were performed. The results showed that ORF24 interacted significantly with all four STM proteins except MAVS. Subsequent fluorescence colocalization experiments further confirmed that ORF24-EGFP exhibited clear colocalization with DsRed-MITA, DsRed-TBK1, DsRed-IRF3, and DsRed-IRF7 in the cytoplasm. These results indicate that ORF24 can directly interact with RLR signaling molecules other than MAVS.

[0025] S6. Finally, confocal imaging is required: 293T cells are seeded on glass coverslips and transfected with plasmids for 24 hours. The cells are then washed twice with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde for 20 minutes, washed three more times with PBS, and stained with 1 mg / mL 4′,6-diamidinyl-2-phenylindole (DAPI, Beyotime) for 15 minutes at room temperature in the dark. Finally, the cells were imaged using a Nikon laser scanning confocal microscope (N-storm; Nikon super-resolution microscope). Imaging was performed using an electron multiplication charge-coupled device (EMCCD) camera (iXonDU-897, Andor) and a 100×, numerical aperture (NA) 1.49 TIRF objective (CFI Apochromat TIRF, Nikon). Fluorescence signals were excited by a 488 nm laser and collected through a bandpass emission filter (500–545 nm). Image acquisition and reconstruction were performed using Nikon NIS-Elements software, and the experimental data were statistically analyzed. The overexpression experiment confirmed that ORF24 had no significant effect on the protein expression of interacting RLR signaling molecules. To further investigate whether ORF24 blocks the IFN signaling pathway, the recruitment effects of MITA and TBK1 on downstream IRF3 / 7 were first examined. The Western blot analysis steps were as follows: overexpressing Myc-IRF3 / HA-IRF7 and Flag-MITA... Cell lysates of either Flag-TBK1 or Flag-IRFA were immunoprecipitated with anti-Flag antibody, followed by detection with anti-Myc or anti-HA antibody. Results showed that Myc-IRF3 / HA-IRF7 were co-precipitated in the presence of Flag-MITA or Flag-TBK1, indicating that both MITA and TBK1 have the ability to recruit IRF3 / 7. Previous studies have confirmed the recruitment ability of MITA and TBK1. This study further investigated the effect of ORF24 overexpression on the interaction between MITA / TBK1 and IRF3 / IRF7. The Western blot analysis procedure was as follows: Cell lysates overexpressing Myc-IRF3 / HA-IRF7, Flag-MITA, or Flag-TBK1 were co-transfected with increasing concentrations of EGFP-ORF24, followed by immunoprecipitation with anti-Flag antibody, and detection with anti-Myc or anti-HA antibody. Results showed that with increasing EGFP-ORF24 concentration, Flag-MITA... The gradual decrease in gray values ​​of Myc-IRF3 / HA-IRF7 precipitated in the Flag-TBK1 group suggests that ORF24 interferes with the recruitment process of IRF3 / 7 by MITA and TBK1 in a dose-dependent manner.

[0026] This study investigated the immune escape mechanism of the early CyHV-3 protein ORF24 in host cells. The results showed that ORF24 inhibits interferon production, thereby promoting viral replication. The results indicated that ORF24 regulates interferon expression through interaction with the classical RLR signaling pathway. Further experiments confirmed that ORF24 inhibits host IFN production by interfering with the recruitment of IRF3 / 7 by MITA and TBK1, thus promoting CyHV-3 replication.

[0027] In the long-term evolutionary interaction between viruses and hosts, a variety of viruses have evolved diverse immune escape strategies to circumvent the host's antiviral defense mechanisms. Typical viral immune escape mechanisms include the following: The P protein of SVCV can directly bind to the host transcription factor STAT1 / 2 and prevent its nuclear translocation. STAT1 / 2 is a key molecule that induces the expression of downstream antiviral genes (such as ISGs) by IFN; the blockage of STAT1 / 2 nuclear translocation will prevent ISGs from being transcribed normally, thereby effectively blocking the host's antiviral state and allowing the virus to escape immune clearance. CyHV-2 achieves immune escape through the protein encoded by ORF67: this protein targets key factors in the RLRs signaling pathway, inhibits the phosphorylation of sting-B by TBK1-A / B, and thus affects the production of interferon. Phosphorylated sting is the core molecule that activates the production of interferon (IFN); its phosphorylation function is impaired, which will directly inhibit IFN synthesis, weaken the host's antiviral immune response, and create favorable conditions for viral replication

[30] . Crucian carp herpesvirus (CaHV) utilizes a protein encoded by ORF56 to target the host mitochondrial antiviral signaling protein MAVS-B, thereby promoting viral immune escape. After binding to MAVS-B, ORF56 recruits the selective autophagy receptor OPTN-B, mediating the autophagic degradation of MAVS-B. MAVS is a key activator of the IFN pathway in fish; its degradation significantly inhibits IFN production, thus promoting viral replication within host cells. In contrast, SVCV employs a unique immune escape mechanism, primarily targeting the JAK-STAT signaling pathway. These strategies, by targeting key nodes in the host immune signaling pathway, inhibit IFN production or signal transduction, demonstrating the high adaptability exhibited by viruses during long-term evolution. Notably, all three viruses regulate immune responses by acting on single molecular targets: SVCV's P protein interferes with downstream IFN signal transduction, while CyHV-2's ORF67 and CaHV's ORF56 target IFN. The upstream activation process works through methods such as "inhibition of modification", "induction of degradation" or "blocking of nuclear localization". Herpes simplex virus type 1 (HSV-1) is a common enveloped large double-stranded DNA virus in the Herpesviridae family that can infect humans. After infection, the virus enters a latent state in infected cells (especially sensory neurons). In addition, the virus also encodes the UL38 protein to mediate immune escape. This protein interacts with the sting protein, thereby blocking the sting-TAK1-IRF3 signaling pathway and inhibiting immune signal transduction.

[0028] The full-length genome of CyHV-3 is 295 kbp and contains a large central unique region with a terminal direct repeat sequence of about 22 kbp on each side. The genome encodes 156 potential open reading frames (ORFs), of which 148 ORFs (ORF9–ORF156) are located in the central unique region and another 8 ORFs (ORF1–ORF8) are located in the terminal direct repeat sequences

[35] . Its core pathogenic mechanism relies on a precise immune escape strategy. On the one hand, the ORF89 protein degrades the host antiviral transcription factor IRF3 through the autophagy pathway, inhibiting its dimerization and nuclear translocation, thereby directly blocking the production of interferon (IFN) and weakening the innate immune response. On this basis, CyHV-3 MCP further inhibits the expression of downstream ISG. On the other hand, viral IL-10 (vIL-10) encoded by ORF134 is highly homologous to host IL-10. It can mimic the function of host IL-10, competitively bind to IL-10 receptors, and inhibit macrophage activation, release of pro-inflammatory cytokines and T cell proliferation, thereby forming an immunosuppressive microenvironment.

[0029] As a conserved gene in the herpesvirus genome, ORF24 exhibits highly differentiated functions in different viruses, and this functional differentiation is closely related to its host adaptation and viral life cycle strategy. For channel catfish herpesvirus (IcHV-1), ORF24 has been clearly identified as an immediate early (IE) gene. The gene encoded by this protein has typical IE gene characteristics: it can be activated by the host transcription system in the early stage of viral infection (in the absence of other viral proteins); and it regulates the temporal expression of subsequent early (E) and late (L) genes by binding to specific cis-acting elements in the viral promoter region

[39] . Most studies focus only on gene classification and preliminary functional screening, while neglecting the dynamic interaction between the virus and its host. In contrast, this study systematically explored the potential mechanisms from phenotype to signaling pathways, focusing on the molecular mechanisms of virus-host interaction. The function of ORF24 in Kaposi's sarcoma-associated herpesvirus (KSHV) differs significantly from that in IcHV-1: The KSHV ORF24 protein mimics the structure and function of the TATA-binding protein (TBP), replacing the host TBP through molecular mimicry to bind to the TATA binding site in the promoter of late viral genes. More importantly, ORF24, along with two other virally encoded proteins—ORF34 (transcription cofactor) and ORF21 (viral RNA polymerase subunit)—forms a stable trimeric transcription activation complex. This complex specifically recognizes the promoter sequences of late-stage KSHV genes, significantly enhancing the recruitment efficiency of RNA polymerase. This drives high-level expression of late-stage structural genes, including viral capsid and envelope proteins, providing the material basis for the assembly of progeny viral particles. This mechanism constitutes a core regulatory step in KSHV lytic replication, directly affecting viral particle yield and infectivity. However, previous studies on KSHV ORF24 did not involve host signaling pathways; these studies only focused on the binding of the viral protein complex to the late-stage viral promoter, confirming only the necessity of this complex for late-stage gene expression without linking it to the overall viral replication phenotype. In contrast, this study focuses on the core RLR signaling pathway, elucidating the complete signal transduction chain encompassing molecular interactions, pathway inhibition, and phenotypic changes. More importantly, the experiments directly reveal the underlying mechanisms behind ORF24 expression, pathway inhibition, reduced IFN production, and enhanced viral replication.

[0030] In summary, this study confirms that the early protein ORF24 of CyHV-3 plays a crucial role in virus-host interactions and immune escape. Experiments show that ORF24 promotes viral immune escape by weakening the ability of MITA and TBK1 to recruit IRF3 / 7 and inhibiting host IFN production. The discovery of the ORF24 immune escape mechanism lays the foundation for future development of anti-CyHV-3 drugs and disease detection methods. Further research will explore the interactions between the various functional domains of ORF24 through further experiments.

[0031] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for promoting viral replication of cheetah herpesvirus through signaling pathway-mediated innate immune escape, characterized by: Its replication method includes the following steps: S1. First, the materials need to be prepared, including cells and viruses: Common carp brain (CCB) cells are cultured in M199 medium supplemented with 10% fetal bovine serum (FBS, Life Technologies) at 28°C and 5% CO2 atmosphere; human embryonic kidney 293T cells are cultured in DMEM medium (Life Technologies) containing 10% FBS at 37°C and 5% CO2 atmosphere. For virus amplification, CCB cells are infected with CyHV-3 virus, and the collected virus samples are stored at -80°C. Reagents and antibodies are also included: negative control (NC), the sequences of NC are 5′-uucuccgaacgugucactt-3′ (forward) and 5′-acgugacacguucggagaat-3′ (reverse); mouse anti-Flag, anti-hemagglutinin (HA), anti-Myc and anti-enhanced green fluorescent protein (EGFP) antibodies; S2. Next, plasmid construction is required: Total RNA was extracted from CyHV-3-infected CCB cells using TRIzol reagent (Invitrogen). The open reading frame (ORF) of ORF24 (GenBank ID: 11266358) was amplified by reverse transcription polymerase chain reaction (RT-PCR) and cloned into the pEGFP-N1 vector. All plasmids were constructed according to the specified method, and all constructs were verified by DNA sequencing. Luciferase activity assay is required: CCB cells were seeded in 24-well plates and cultured for 12 hours. Subsequently, plasmids expressing MAVS, TBK1, MITA, IRF3, or IRF7 were combined with IFNφ1pro-Luc, IFNφ2pro-Luc, or IFNφ3pro-Luc. - Luc or ISREpro-Luc reporter plasmid (including pRL-TK Renilla luciferase plasmid as an internal control) were co-transfected into cells. 24 hours after transfection, cells were washed with phosphate-buffered saline (PBS), and luciferase activity was measured using the Dual-Luciferase Reporter Assay Kit (YEASEN) according to the manufacturer's instructions. To ensure the accuracy of subsequent data analysis, firefly luciferase activity was standardized to Renilla luciferase activity. S3. Next, transfection is required. Cells in the 12-well plate are transfected using Trans-Introduction. Transfection was performed using EL transfection reagent (TransGen Biotech) in 200 μL of optimized serum-free medium. Six hours post-transfection, the medium was replaced with 1 mL of M199 medium or Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Cells were then cultured for another 18 hours before viral infection experiments: CCB cells were inoculated with CyHV-3 virus at a multiplicity of infection (MOI) of 10. Control cells were simultaneously treated with an equal volume of phosphate-buffered saline (PBS). All cells were then cultured according to the same protocol, and relevant indicators were measured. After adsorption at 25°C for 2 hours, the inoculum was removed, and the medium was replaced with M199 medium containing 5% fetal bovine serum (FBS). Cells were collected at 12, 24, 36, and 48 hours post-infection, and viral mRNA levels were detected using quantitative real-time polymerase chain reaction (qRT-PCR). S4. Next, RNA extraction, reverse transcription, and qRT-PCR are required. Total RNA is extracted using TRIzol reagent. PrimeScript is used according to the manufacturer's instructions. First-strand complementary DNA (cDNA) was synthesized using an RT kit (containing gDNA Eraser, Takara Bio). Quantitative real-time PCR (qRT-PCR) was performed on a CFX96 real-time quantitative PCR system (Bio-Rad) using AceQ. qPCR SYBR Green premix (Vazyme Biotech) is used for Western blot analysis: Total cellular proteins are extracted using cell lysis buffer (containing 50 mM Tris [pH 7.4], 150 mM sodium chloride, 1% Triton X-100, 1% sodium deoxycholate, and 0.1% SDS). Protein samples are denatured at 100°C for 10 minutes, separated by 10% SDS-PAGE gel electrophoresis, and then transferred to a nitrocellulose membrane (Biosharp). The membrane is blocked overnight at 4°C in Tris-buffered saline (TBST) containing 5% skim milk powder, followed by incubation with the specified primary antibody (Abs) at room temperature for 2 hours. After washing three times with TBST, the membrane is then incubated with IRDye at room temperature. Incubation with 800CW-labeled donkey anti-rabbit IgG (1:10,000) or anti-mouse IgG (1:10,000) for 1 hour, signal intensity was detected using an Amersham Imager 680 (Cytiva); S5. The co-immunoprecipitation experiment needs to be performed in sequence: Seed an appropriate amount of cells into a culture flask and culture until the cell density reaches 70-80% confluence. Then, transfect the cells with a total of 16 μg of target plasmid. After culturing for 24 hours, add 2 mL of ice-cold phosphate-buffered saline (PBS), gently wash the cell monolayer twice, scrape the cells off the culture flask wall with a cell scraper, collect the cell suspension and transfer it to a centrifuge tube. Centrifuge at 1000 rpm for 5 minutes and collect the cell pellet. Add pre-chilled PBS to the pellet, gently resuspend the cells using a pipette, and centrifuge again at 1000 rpm for 3 minutes. Discard the supernatant to obtain a purified cell pellet. Add an equal volume of lysis buffer (Sigma-Aldrich, St. Louis, Missouri, USA) to the pellet volume, mix the cells thoroughly by repeated pipetting, and incubate on ice for 10–20 minutes to achieve complete lysis. Transfer the lysis buffer to a centrifuge tube and centrifuge at 12,000 × g for 15 minutes at 4°C. After centrifugation, collect the supernatant containing soluble protein, discard the cell debris at the bottom, and transfer the collected protein supernatant to a new centrifuge tube. Add 30 μL of anti-Flag affinity gel (DIA-AN), place the centrifuge tube in a shaker, and gently vortex overnight at 4°C to allow the Flag-labeled protein to fully bind to the affinity gel. After incubation, collect the affinity gel bound to the target protein by centrifuging at 5000 × g for 3 minutes at 4°C and discard the supernatant. Add an appropriate amount of ice-cold PBS to the gel, gently resuspend and centrifuge again, discard the supernatant, repeat this washing step three times to remove non-specifically bound contaminating proteins, add 25 μL of 5×SDS sample buffer to the washed affinity gel, resuspend and mix thoroughly to obtain immunoprecipitated protein samples, and perform Western blot (IB) analysis on the samples after subsequent processing to further detect proteins. S6. Finally, confocal imaging is required: 293T cells are seeded on glass coverslips and transfected with plasmids for 24 hours. The cells are then washed twice with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde for 20 minutes, washed three more times with PBS, and stained with 1 mg / mL 4′,6-diamidinyl-2-phenylindole (DAPI, Beyotime) for 15 minutes at room temperature in the dark. Finally, the cells were imaged using a Nikon laser scanning confocal microscope (N-storm; Nikon super-resolution microscope). Imaging was performed using an electron multiplication charge-coupled device (EMCCD) camera (iXon DU-897, Andor) and a 100×, numerical aperture (NA) 1.49 TIRF objective lens (CFI Apochromat TIRF, Nikon). Fluorescence signals were excited by a 488 nm laser and collected through a bandpass emission filter (500–545 nm). Image acquisition and reconstruction were performed using Nikon NIS-Elements software, and the experimental data were statistically analyzed.

2. The method for promoting viral replication of cheetah herpesvirus through signaling pathway-mediated innate immune escape according to claim 1, characterized in that: The plasmids pCDNA-MAVS (NM_001080584.2), pCMV-Tag2C-MAVS, pCDNA-TBK1 (NM_001044748.2), pCMV-Tag2C-TBK1, pD sRed-TBK1, pCDNA-MITA (NM_001278837.1), pCMV-Tag2C-MITA, pDsRed-MITA, pCDNA-IRF3 (NM_001143904), pCM V-Tag2C-IRF3, pDsRed-IRF3, pCDNA-IRF7 (NM_200677.2), pCMV-Tag2C-IRF7, pDsRed-IRF7, as well as IFNφ1pro-Luc, IFNφ2pro-Luc, IFNφ3pro-Luc, interferon-stimulated response element (ISRE)-Luc, pMyc-IRF3, and pHA-IRF7 were constructed according to the previously described method, and all constructs were validated by DNA sequencing.

3. The method for promoting viral replication of cheetah herpesvirus through signaling pathway-mediated innate immune escape according to claim 1, characterized in that: The formula for calculating the standardized luciferase activity is as follows: Standardized luciferase activity = Firefly luciferase reading ÷ Corresponding Renida luciferase reading.

4. The method for promoting viral replication of cheetah herpesvirus through signaling pathway-mediated innate immune escape according to claim 1, characterized in that: The PCR cycling conditions were as follows: initial denaturation at 95°C for 5 minutes; followed by 40 cycles, each cycle consisting of denaturation at 95°C for 10 seconds, annealing at 60°C for 10 seconds, and extension at 72°C for 15 seconds. After amplification, melting curve analysis was performed at 95°C with a heating rate of 5°C / s to verify the specificity of the PCR products. - The ΔΔCt method (double ΔCt method) normalizes the data of each sample to the level of β-actin (internal reference gene).

5. The method for promoting viral replication of cheetah herpesvirus through signaling pathway-mediated innate immune escape according to claim 1, characterized in that: All statistical analyses were performed using GraphPad Prism 5.0 (GraphPad Software). Statistical significance was determined by a two-tailed unpaired Student's t-test, with p < 0.05 considered statistically significant. All data represent the results of at least two independent experiments, each repeated three times (mean ± standard deviation). An asterisk (*) indicates a statistically significant difference (*p < 0.05, **p < 0.01).

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