Application of PCV2 in preparation of oncolytic virus

Abstract

The invention provides application of PCV2 in preparation of oncolytic viruses, and belongs to the technical field of biological medicines. The invention proposes that PCV2 can be used as an oncolytic virus for the first time, the PCV2 can efficiently infect various human tumor cell lines in a broad-spectrum manner, replication and filial generation virus assembly are completed in cytoplasm, and finally virus particles are released in a cell lysis or apoptosis way. The novel oncolytic virus PCV2 provided by the invention is low in human normal cell tropism and has natural tumor cell tropism. Compared with the traditional virulent oncolytic virus, the PCV2 has a mild killing effect, and mostly realizes tumor depletion death by inhibiting cell growth, limiting tumor energy supply, inhibiting tumor cell migration and invasion and other anti-tumor effects, so that the PCV2 is safer. Through xenotransplantation tumor-bearing mouse model in-vivo experiments, the PCV2 shows an obvious oncolytic effect in lung cancer and cervical cancer, and has an obvious clinical application value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of PCV2 in the preparation of oncolytic viruses. Background Technology

[0002] Oncolytic viruses (OVs), as an emerging cancer treatment strategy, have shown great promise in basic research and clinical translation in recent years through a dual mechanism of selectively infecting and lysing tumor cells and activating anti-tumor immune responses. Their core principle lies in utilizing the biological contradiction between the natural replication characteristics of viruses and the unique microenvironmental defects of tumor cells (such as inactivation of antiviral signaling pathways and abnormal cell cycle regulation) to achieve tumor-specific killing while simultaneously reshaping the immunosuppressive microenvironment. This "two birds with one stone" treatment concept breaks through the limitations of traditional radiotherapy and chemotherapy, becoming an important branch of cancer immunotherapy.

[0003] Despite the immense potential of oncolytic virus therapy in cancer treatment, existing viral vectors (such as adenovirus, herpesvirus, and vaccinia virus) still face numerous technical bottlenecks. First, after systemic administration, viral particles are easily cleared by complement, neutralizing antibodies, and phagocytes in the blood, significantly reducing the actual delivery efficiency to the tumor site. Second, since most candidate viruses originate from common human pathogens (such as adenovirus and measles virus), pre-existing immunity in the population further weakens the virus's ability to infect and replicate in tumor tissues, severely impacting treatment efficacy. Furthermore, gene modifications introduced to enhance tumor specificity (such as deleting viral virulence genes or inserting tumor-specific promoters) often weaken the virus's replication efficiency, and complex engineering designs significantly increase manufacturing processes and quality control costs.

[0004] Porcine circovirus type 2 (PCV2) is a pathogen that primarily infects pigs. It is a small, non-enveloped, single-stranded DNA virus with a genome length of approximately 1.7 kb, encoding replication-associated proteins (Rep) and capsid proteins (Cap). There are currently no reports in this field regarding the use of PCV2 as an oncolytic virus. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide the application of PCV2 in the preparation of oncolytic viruses. As an oncolytic virus, PCV2 can broadly and efficiently infect a variety of human cancer cells. After infection, it can cause tumor cell cycle arrest, growth restriction, inhibition of tumor cell energy supply, inhibition of tumor cell migration and invasion, and other anti-tumor effects, thereby achieving the purpose of tumor consumption death.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the application of PCV2 in the preparation of oncolytic viruses.

[0007] This invention also provides the application of PCV2 in the preparation of tumor treatment products.

[0008] Preferably, the nucleotide sequence of the PCV2 is shown in SEQ ID NO.1.

[0009] Preferably, the tumor includes lung cancer, cervical cancer, liver cancer, pancreatic cancer, breast cancer, lymphoma, or monocytic leukemia.

[0010] This invention also provides the application of PCV2 in the preparation of products that promote tumor cell apoptosis, inhibit tumor cell migration, or inhibit tumor cell invasion.

[0011] Preferably, the tumor cells include human lung cancer cells A549, human cervical cancer cells HeLa, human liver cancer cells HepG2, human pancreatic cancer cells PANC-1, human breast cancer cells MCF-7, lymphoma U937, or monocytic leukemia THP-1.

[0012] The present invention also provides an oncolytic virus, including PCV2, wherein the effective dose of PCV2 is 1~10×10 6 TCID 50 / mL.

[0013] This invention also provides the application of the above-mentioned oncolytic virus in the preparation of tumor treatment products.

[0014] The beneficial effects of this invention are: This invention proposes for the first time that PCV2 can be used as an oncolytic virus. PCV2 can broadly and efficiently infect various human tumor cell lines, completing replication and progeny virus assembly in the cytoplasm, and ultimately releasing viral particles through cell lysis or apoptosis. The novel oncolytic virus PCV2 provided by this invention can broadly and efficiently infect tumor cells, while exhibiting weak tropism for normal human cells and possessing natural tumor cell tropism. Compared with traditional virulent oncolytic viruses, PCV2 has a milder killing effect, primarily through inhibiting cell growth and limiting tumor energy supply, inhibiting tumor cell migration and invasion, and achieving tumor-depleting cell death, thus demonstrating greater safety. In in vivo experiments using xenograft tumor-bearing mouse models, PCV2 showed significant oncolytic effects in both lung cancer (A549) and cervical cancer (HeLa), demonstrating significant clinical application value. Attached Figure Description

[0015] Figure 1To indirectly identify the effectiveness of PCV2 in infecting various human cells using immunofluorescence, green fluorescence represents a PCV2 Cap-specific positive fluorescence signal, and blue fluorescence represents DAPI-stained cell nuclei. Figure 2 The average fluorescence intensity of PCV2 Cap positive signal in different cell lines; Figure 3 Growth curves of PCV2 in different cell lines; Figure 4 To compare the cell states of different cells infected and uninfected with PCV2 in the F1, F3 and F5 generations, the images were taken with the adherent cells in sharp focus. The white dots in the images are suspended dead cells or cell debris. Figure 5 PCV2 infection of A549 cells promotes the accumulation of p53; Figure 6 The effects of PCV2 infection of A549 cells and overexpression of PCV2 Cap and Rep proteins on the cell cycle were investigated. This indicates that p < 0.001; Figure 7 The effect of PCV2 infection on pyruvate kinase activity in human cells is given by ns, where ns indicates no statistical significance (p>0.05). This indicates that p < 0.001; Figure 8 The effect of PCV2 infection on ATP production in human cells is represented by ns, where ns indicates no statistical significance (p>0.05). This indicates that p < 0.001; Figure 9 Color comparison of tumor cells inoculated with PCV2 and those not inoculated with PCV2 after 24 hours of cell culture; Figure 10 To determine the effect of PCV2 infection on lactate production in human cells, ns indicates no statistical significance (p>0.05). This indicates that p < 0.001; Figure 11 The results of PCV2 inhibiting tumor cell migration and invasion are shown in Figure A, which is the cell scratch assay result, mainly comparing the scratch closure rate (i.e., the area between the red lines). The faster the scratch closure rate (i.e., the smaller the area between the red lines at a certain time point), the stronger the cell migration ability. Figure B is the Transwell chamber cell invasion assay result, comparing the number of cells that penetrate into the lower chamber. The more cells that penetrate into the lower chamber, the stronger the invasion ability. Figure 12 A diagram illustrating subcutaneous tumor tissue from BALB / c nude mice inoculated with human lung cancer cells (A549 cells); Figure 13Comparison of subcutaneous tumor sizes in mice inoculated with human lung cancer cells (A549 cells) using BALB / C nude mice; Figure 14 A comparison of subcutaneous tumor weight in mice inoculated with human lung cancer cells (A549 cells) using BALB / c nude. This indicates that p < 0.001; Figure 15 The results are used to identify the PCV2 infection status in tumor tissue. The top row of images is a scan of the entire tumor tissue, and the bottom row is a magnified view of the local area. Brown signals represent PCV2 positive signals, and the scale bar in the images is 100nm. Figure 16 The images show a comparison of HE staining of tumor tissues from the placebo group and the PCV2 group. The top row shows a panoramic scan of HE staining of tumor tissues from the placebo group and the PCV2 group, and the bottom row shows a magnified view of the area within the red box. Figure 17 The image shows a comparison of TUNEL staining in tumor tissues from the placebo group and the PCV2 group. Red fluorescence indicates apoptosis, and blue fluorescence indicates the location of cell nuclei stained with DAPI. The image below is a magnified view of the part in red in the image above. Figure 18 To observe the invasion and metastasis of tumor cells in tumor tissue sections, the blue arrows indicate the mouse muscle tissue layer, and the red arrows indicate new metastatic lesions. Figure 19 Image showing the organs of a dissected mouse; Figure 20 A diagram illustrating subcutaneous tumor tissue from BALB / C nude mice inoculated with human cervical cancer cells (HeLa cells); Figure 21 Growth curve of subcutaneous tumors in BALB / C nude mice inoculated with HeLa cells; Figure 22 A comparison of subcutaneous tumor weight in BALB / C nude mice after HeLa cell inoculation. This means p < 0.01. Detailed Implementation

[0016] This invention provides the application of PCV2 in the preparation of oncolytic viruses.

[0017] In this invention, when PCV2 is used as an oncolytic virus, it has the following significant transformational advantages: 1. Low pre-existing immune barrier: Humans have no history of natural infection with PCV2, and the positive rate of specific neutralizing antibodies in serological surveys is extremely low (<5%), which can avoid the interference of pre-existing immunity on virus delivery; 2. Excellent safety characteristics: Although PCV2 is pathogenic to pigs, it only causes subclinical infection in humans, with no clear reports of pathogenicity, and its genome does not contain known oncogenes or strong immune escape modules; 3. Inherent immune activation potential: PCV2 infection can induce endoplasmic reticulum stress and mitochondrial apoptosis in tumor cells, while upregulating the expression of MHC-I / II molecules on the cell surface, promoting dendritic cell maturation and T cell activation; 4. Structural stability and ease of modification: Its small genome and simple structure are conducive to genetic engineering modification (such as inserting immunomodulatory factor genes), and its non-enveloped nature makes it naturally resistant to complement attack in the blood. In this invention, given PCV2's natural targeting of human tumor cells, its biological characteristics of effectively promoting apoptosis and autophagy in infected cells, its extremely low pre-existing immunity, and its excellent safety (common subclinical infection and naturally non-pathogenic to humans), a unique foundation has been laid for its transformation into an oncolytic virus.

[0018] This invention also provides the application of PCV2 in the preparation of tumor treatment products.

[0019] The PCV2 provided in this invention, as a novel oncolytic virus, exhibits virological characteristics. Its genome consists of only 1.76 kb of closed circular DNA, and its replication depends on the host cell's DNA polymerase. This characteristic makes PCV2 preferentially infect highly proliferating cells (such as rapidly dividing lymphocytes and epithelial cells), a feature highly compatible with the rapid division of tumor cells. This precisely enhances PCV2's specificity for infecting tumor cells while reducing toxicity to normal cells. Furthermore, from a viral pathogenesis perspective, PCV2 Cap can trigger various apoptotic pathways, and the PCV2-induced apoptosis process can be further enhanced in IFN-γ-treated cells. This characteristic may be amplified in the tumor microenvironment. Notably, in addition to inducing apoptosis, PCV2 also induces autophagy, thus PCV2 infection has a strong ability to induce tumor cell apoptosis and autophagy. This provides the fundamental conditions for PCV2 to become an oncolytic virus. At the level of immune regulation, PCV2 infection significantly upregulates the expression of pro-inflammatory factors such as IL-6, IL-8, and TNF-α, while inducing MHC-I molecules to present viral antigens, thereby activating CD8 through antigen cross-presentation. +T cells may disrupt the immunosuppressive tumor microenvironment. Furthermore, the circular, plasmid-like genome of PCV2 facilitates genetic modification. The viral DNA is also more stable within cells and less likely to integrate into the cellular genome, offering a degree of safety. Additionally, PCV2 infection in pigs is mostly subclinical, with infected pigs remaining carriers for extended periods. This demonstrates that PCV2 possesses a complex and efficient immune escape mechanism, failing to trigger a strong viral immune response, thus ensuring the virus can remain in the body long-term and exert its oncolytic effect. Moreover, as an animal virus, PCV2 has a natural safety advantage for cross-species applications due to low pre-existing human immunity and weak pathogenicity.

[0020] In this invention, the preferred nucleotide sequence of PCV2 is shown in SEQ ID NO.1. This invention does not specifically limit the source of PCV2; it can be obtained by self-isolation and propagation from PCV2-positive pig tissues, or by in vitro synthesis based on the nucleotide sequence. In this invention, after obtaining PCV2, it is preferably amplified in PK15 cells, and purified and concentrated using ultracentrifugation, then aliquoted to obtain PCV2 virus stock solution for later use.

[0021] In this invention, the tumor preferably includes lung cancer, cervical cancer, liver cancer, pancreatic cancer, breast cancer, lymphoma, or monocytic leukemia.

[0022] This invention also provides the application of PCV2 in the preparation of products that promote tumor cell apoptosis, inhibit tumor cell migration, or inhibit tumor cell invasion.

[0023] In this invention, the tumor cells preferably include human lung cancer cells A549, human cervical cancer cells HeLa, human liver cancer cells HepG2, human pancreatic cancer cells PANC-1, human breast cancer cells MCF-7, lymphoma U937, or monocytic leukemia THP-1. This invention does not specifically limit the origin of the above cell lines.

[0024] The present invention also provides an oncolytic virus, including PCV2, wherein the effective dose of PCV2 is 1~10×10 6 TCID 50 / mL, the preferred nucleotide sequence of the PCV2 is shown in SEQ ID NO.1.

[0025] This invention also provides the application of the above-mentioned oncolytic virus in the preparation of tumor treatment products.

[0026] In this invention, the product preferably includes a drug.

[0027] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0028] Unless otherwise specified, the following embodiments are all conventional methods.

[0029] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0030] Example 1 Amplification, purification, and concentration of novel oncolytic virus PCV2 in PK-15 cells: Step 1: Culture and preparation of PK-15 cells: 1. Cell resuscitation and expansion: Resuscitate frozen PK-15 cells and culture them in T75 flasks in DMEM high-glucose medium containing 10% FBS and 1% penicillin antibiotics. Incubate at 37°C in a 5% CO2 incubator until confluence reaches 80-90%. Passage cells regularly (every three days) to maintain optimal cell growth. Expand a sufficient number of cells for viral infection.

[0031] 2. Cell inoculation: The day before infection, cells were digested with trypsin and counted. PK-15 cells were then packed at approximately 5 × 10⁶ cells per cell. 5 Cells / mL were seeded into multiple T175 culture flasks, and each flask was supplemented with sufficient DMEM high-glucose medium. Cell confluence was increased to 70-80% by the second day.

[0032] Step Two: Virus Inoculation and Amplification 1. Virus adsorption: Remove the cell culture flask and discard the old culture medium. Gently wash the cell surface twice with pre-warmed sterile PBS to remove any residual serum. Calculate the required volume of PCV2 virus solution (here, PCV2 virus solution is the stock solution of virus from lung tissue of PCV2-positive pigs, which has been ground, filtered, inoculated into PK15, and propagated before preservation; the nucleotide sequence of PCV2 in the PCV2 virus solution is shown in SEQ ID NO.1) according to the predetermined multiplicity of infection (MOI=1.0). Dilute the virus solution with a small amount of serum-free or low-serum (2% FBS) DMEM high-glucose medium, mix well, and add to the culture flask, ensuring that the liquid evenly covers the cell layer. Incubate at 37°C for 2 hours for adsorption, gently shaking the flask every 15 minutes to ensure even distribution of the virus.

[0033] 2. Replace the maintenance medium and continue culturing: After adsorption is complete, discard the virus inoculum. Add fresh maintenance medium containing 2% FBS and 1% penicillin antibody (e.g., 35 mL / T175 flask). Return to a 37°C incubator for further incubation. Incubate for 3-5 days to ensure sufficient virus replication.

[0034] Step 3: Virus harvesting; The cell culture flasks were repeatedly freeze-thawed three times (frozen at -80°C and thawed in a 37°C water bath) to release the virus from the cells. Cell debris was removed by centrifugation at 5000 rpm for 20 minutes at 4°C. The supernatant was collected; this is the crude virus solution. It can be aliquoted and stored at -80°C or purified immediately.

[0035] Step 4: Virus purification and concentration (ultracentrifugation): 1. Sucrose density gradient centrifugation: Prepare the ultracentrifuge tubes. First, slowly add 2-3 mL of 20% sucrose pad solution to the bottom of the tube. Then, very carefully and slowly add the crude virus solution on top of the sucrose solution, avoiding interface mixing, until it is about 3-5 mm from the top of the tube. Accurately level the centrifuge tubes (weight difference <0.01 g). Using an ultracentrifuge (such as a Beckman Coulter Optima XPN series), select an SW 32Ti rotor (or equivalent rotor) and centrifuge at 100,000-150,000 × g for 2-4 hours at 4°C. After centrifugation, the virus particles will form a milky white, translucent viral band on the sucrose pad.

[0036] 2. Collect virus bands: Carefully remove the centrifuge tube, avoiding any vibration. Using a sterile syringe needle, carefully insert it into the side of the tube, aiming at the viral band, and slowly aspirate the virus-containing liquid. Transfer the collected viral fluid to a new centrifuge tube.

[0037] 3. Dialysis or dilution to remove sugar: The collected virus fluid and sucrose mixture was dialyzed overnight (4°C) with a large amount of pre-cooled PBS, or diluted and concentrated using an ultrafiltration tube to remove the sucrose.

[0038] Step 5: Titer determination (TCID) 50 Law): Serial dilution: The purified and concentrated virus stock solution was serially diluted 10-fold in DMEM medium containing 2% FBS and high glucose (from 10... -1 Up to 10 -8 (or higher).

[0039] Seeding in 96-well plates: A monolayer of PK-15 cells (confluence greater than 90%) has been pre-seeded in the 96-well plates. Discard the old culture medium from each well. Seed 8-10 replicate wells for each dilution, adding 100 μL of diluted virus solution to each well. Include control wells with only maintenance medium. Incubate the 96-well plates at 37°C in a 5% CO2 incubator for 3 days.

[0040] Indirect immunofluorescence staining: Fixation: After incubation, discard the culture medium. Add 100 μL of pre-cooled ethanol:acetone (1:1) fixative to each well and fix for 10-15 minutes at room temperature. Discard the fixative and allow the plate to air dry in a ventilated area for 15-30 minutes (acetone and ethanol will evaporate).

[0041] Breakthrough: Treat with PBS containing 0.1%~0.5% Triton X-100 at room temperature for 10 minutes, then wash 3 times with PBS.

[0042] Blocking: Add 100-200 μL of 1-5% BSA blocking solution to each well and block at room temperature for 30 minutes. After blocking, discard the blocking solution; no washing is required.

[0043] Add primary antibody: Dilute mouse anti-PCV2 antibody (GeneTex) to working concentration (1:1000) with antibody dilution buffer (1% BSA / PBS). Add 50 μL of the diluted primary antibody to each well. Place the cell plate in a humidified chamber and incubate at 37°C for 1 hour (or 4°C overnight). After incubation, wash three times with PBS for 5 minutes each time, gently shaking.

[0044] Add secondary antibody: Dilute the FITC-labeled goat anti-mouse IgG antibody with antibody dilution buffer (protect from light), following the manufacturer's recommended dilution ratio (e.g., 1:200~1:1000). Add 100 μL of the diluted secondary antibody to each well (protect from light throughout). Place in a humidified chamber and incubate at 37°C in the dark for 45 minutes to 1 hour. After incubation, wash three times with PBS for 5 minutes each time, protected from light.

[0045] Observation and Counting: A small amount of PBS or mounting medium can be added directly to the plate and observed under a fluorescence microscope. The TCID of the virus is calculated using the Karber formula. 50 / mL.

[0046] Step Six: Packaging and Storage Virus titer greater than 10 6 TCID 50 The titer is set at 100 μL / mL. Aliquot the determined virus solution according to the usage volume (e.g., 100 μL / vial). Clearly label the virus name, titer, preparation date, and other information. Store long-term in an ultra-low temperature freezer at -80°C, avoiding repeated freeze-thaw cycles.

[0047] Example 2 PCV2 can effectively infect and promote tumor cell death and inhibit tumor cell growth. 1. Verify the infection tropism of PCV2 on human cells. To verify the ability of PCV2 to infect human cells across species and its cell tropism, the present invention designed and implemented the following experiments: Several representative human cell lines were selected for testing, including: human tumor cell lines: human lung cancer cells (A549), human cervical cancer cells (HeLa), human liver cancer cells (HepG2), human pancreatic cancer cells (PANC-1), and human breast cancer cells (MCF-7); and human normal cell lines: human embryonic kidney cells (HEK-293T) and human normal bronchial epithelial cells (BEAS-2B). Meanwhile, porcine kidney epithelial cells (PK-15), which are highly sensitive to PCV2, were used as a positive control.

[0048] The PCV2 prepared in Example 1 (nucleotide sequence as shown in SEQ ID NO.1) was used to infect the above cell lines at a multiplicity of infection (MOI=1.0), and then indirect immunofluorescence identification was performed to evaluate the infection efficiency of PCV2 in different cells.

[0049] The results are as follows Figure 1 and Figure 2 As shown, PCV2 effectively infected all tested human cell lines, including tumor and normal cell lines, demonstrating its ability to infect human cells across species. Notably, PCV2 showed significantly higher infection efficiency in human tumor cell lines than in normal human cell lines, exhibiting superior tropism for tumor cells. Specifically, PCV2 demonstrated the highest infection efficiency in human lung cancer cells A549, almost reaching the same level of infection as in the positive control PK-15 cells. This indicates that the A549 cell line is a highly susceptible human cell model for PCV2.

[0050] 2. Analysis of PCV2 viral proliferation kinetics in different human cell lines To clarify the replication characteristics of PCV2 in human cells, this invention further investigated the viral proliferation kinetics in different cell lines. The specific methods are as follows: Human tumor cell lines (A549, HeLa, HepG2, PANC-1, MCF-7) and normal cell lines (HEK-293T, BEAS-2B) were infected with an MOI of 1, with porcine kidney epithelial cells (PK-15) highly sensitive to PCV2 serving as a positive control. Cell samples were collected at 12h, 24h, 36h, 48h, 60h, and 72h post-infection. Total nucleic acid was extracted, and real-time quantitative PCR (qPCR) was used to accurately detect the dynamic changes in PCV2 genome copy number.

[0051] The results are as follows Figure 3As shown, in all tested human tumor cell lines, the PCV2 genome copy number showed a significant upward trend over time, indicating that the virus can complete efficient intracellular replication and proliferation cycles. In contrast, in the two normal human cell lines (HEK-293T and BEAS-2B), the PCV2 nucleic acid content tended to stabilize in the later stages of infection without significant increase, suggesting that although the virus can enter normal human cells, its replication efficiency is low or the replication process is inhibited.

[0052] These results demonstrate that PCV2 can infect human cells and exhibits significant tumor cell selectivity in its replication, meaning it has the potential for efficient replication in human tumor cells while replication is restricted in normal cells. This characteristic further confirms PCV2's significant tropism for human tumor cells, providing crucial theoretical support for its potential as a candidate oncolytic virus strain targeting tumors.

[0053] 3. Effects of PCV2 infection on human cell viability and long-term passage toxicity assessment To evaluate the potential toxic effects of PCV2 on human cells and its impact on persistent infection, this invention conducted multiple passage culture experiments on PCV2-infected cells. Using human normal bronchial epithelial cells BEAS-2B, human lung cancer cells A549, and human cervical cancer cells HeLa as models, a virus-infected group (PCV2 infection, MOI=1) and an uninfected parallel control group (NC) were established, and cells in each group were continuously passaged. The cell morphology, growth status, and survival of the first (F1), third (F3), and fifth (F5) generations were systematically observed.

[0054] The results are as follows Figure 4 As shown, no typical cytopathic effect (CPE) was observed in any of the cell groups throughout the passage period. However, compared to the control group, the PCV2-infected tumor cells (A549 and HeLa) exhibited a significant slowdown in proliferation rate with increasing passage number, and a significant increase in suspended dead cell debris in the culture medium, indicating a deterioration in their survival status and an increase in dead cells. In contrast, the PCV2-infected normal BEAS-2B cells did not show significant growth inhibition or morphological abnormalities at any passage, and their condition was basically consistent with that of the uninfected control group. This cell type difference in toxicity is consistent with the aforementioned finding that PCV2 has a strong replication capacity in tumor cells but limited replication in normal cells.

[0055] The above results indicate that PCV2 infection can specifically inhibit the growth of human tumor cells and promote their death, but no obvious toxic effects were observed on normal human cells. This further confirms that PCV2 has selective tropism and tumor-suppressing effects on human tumor cells, and has the potential to be developed into a novel oncolytic virus.

[0056] Example 3 Effects of PCV2 infection on p53 expression and cell cycle in A549 cells: P53 is one of the most important tumor suppressor genes in the human body, often referred to as the "guardian of the genome." Under normal circumstances, it monitors the health and integrity of cells. However, in tumors, more than 50% of P53 genes mutate and become inactive, turning it from a "guardian" into an "accomplice," leading to cell cycle disorder and thus promoting tumor development and progression.

[0057] To clarify the effect of PCV2 infection on the expression level of P53 protein in A549 cells, cells were treated with PCV2 virus solutions of different multiplicity of infection (MOI), and the accumulation of P53 was detected. The specific experimental steps are as follows: Cell culture and grouping: A549 cells in good growth condition were seeded at an appropriate density into 6-well plates and cultured at 37°C in a 5% CO2 incubator until confluence reached approximately 70%. Virus inoculation: Discard the original culture medium and gently wash the cells twice with PBS. The experimental groups were given PCV2 virus solution obtained in Example 1 with different MOIs (0.1, 0.2, 0.5, 1 and 2), while the control group was given an equal volume of virus-free maintenance culture medium. Culture and collection: After culturing at 37°C for 36 hours, the supernatant was discarded, the cells were washed twice with pre-cooled PBS, cell lysis buffer was added, and the cells were lysed on ice for 30 minutes to collect whole-cell protein samples. Protein detection: Protein concentration was determined by BCA method, and the expression level of P53 protein was detected by Western blotting. GAPDH was used as an internal control for standardization.

[0058] The results are as follows Figure 5 As shown, compared with the control group, the expression level of P53 protein in PCV2-infected A549 cells was significantly increased, and this accumulation phenomenon showed a clear dose-dependent effect: that is, as the PCV2 infection multiple (MOI) increased, the degree of accumulation of P53 protein in the cells increased accordingly.

[0059] To investigate the effects of PCV2 infection and its encoded proteins Cap and Rep on the cell cycle, cell cycle distribution in treated A549 cells was analyzed by flow cytometry. The specific experimental steps are as follows: Cell culture and grouping: A549 cells were seeded in 6-well plates, and experiments were conducted when the cell density reached approximately 70% confluence. The experiment was divided into four groups: blank control group (NC group), PCV2 virus infection group (MOI=1) (PCV2 group), pCDH-Cap overexpression group (PCV2 Cap group), and pCDH-Rep overexpression group (PCV2 Rep group). Viral infection and transfection: PCV2 virus infection group: Discard the original culture medium and add maintenance culture medium containing PCV2 (MOI=1); Overexpression group: using Lipofectamine TM 3000 were transfected with pCDH-Cap and pCDH-Rep plasmids (two recombinant plasmids were obtained by gene synthesis from Wuhan Tianyi Huayu Gene Technology Co., Ltd.), and the operation was performed according to the instructions. The control group was given an equal volume of PBS; Cell culture and collection: Each group of cells was cultured at 37°C and 5% CO2 for another 36 hours. Cells were collected by digestion with trypsin, washed twice with pre-cooled PBS, and then fixed overnight with 70% ethanol at -20°C. Flow cytometry detection: Remove the fixative, resuspend and wash with PBS; Add RNase A (50 μg / mL) and treat for 30 minutes, then add propidium iodide (PI, 50 μg / mL) and stain for 15 minutes under light-protected conditions; Cell cycle distribution was detected using a flow cytometer (such as BD FACSCalibur), with no fewer than 10,000 cells collected in each group, and cycle fitting analysis was performed using ModFit LT software.

[0060] The results are as follows Figure 6 As shown, compared with the control group, the proportion of S-phase cells in the PCV2 infection group, Cap overexpression group and Rep overexpression group was significantly increased (p<0.001), indicating that the expression of PCV2 virus and its Cap or Rep protein alone can induce S-phase arrest in A549 cells.

[0061] These results suggest that PCV2 infection and Cap or Rep proteins are sufficient to induce S-phase arrest in cells. This effect is consistent with the cell cycle regulation caused by the accumulation of P53 protein in cells, further indicating that PCV2 may mediate S-phase arrest by regulating the P53 signaling pathway.

[0062] Example 4 PCV2 inhibits the energy supply from glycolysis in tumor cells and the accumulation of lactic acid, a metabolic product. In most tumor cells, even with sufficient oxygen, they tend to convert large amounts of glucose into lactic acid through glycolysis rather than allowing pyruvate to enter the mitochondria. This phenomenon of high-speed glycolysis and lactic acid production even under sufficient oxygen conditions is called the Warburg Effect.

[0063] The Warburg effect brings the following benefits to the occurrence and development of tumors: Rapid ATP production: Although inefficient, it is extremely fast and can meet the basic needs of cell membrane ion pumps.

[0064] Providing precursors for biosynthesis: This is the most crucial role. Intermediate metabolites produced by rapid glycolysis (such as glucose-6-phosphate, 3-phosphoglycerate, etc.) can be diverted to the pentose phosphate pathway, amino acid synthesis pathway, and lipid synthesis pathway, providing raw materials for DNA replication, protein synthesis, and cell membrane construction.

[0065] Maintaining the microenvironment: Producing lactic acid, acidifying the microenvironment, which helps tumor invasion and immune escape.

[0066] To clarify whether PCV2 regulates tumor energy metabolism by affecting the enzymatic activity of pyruvate kinase M2 (PKM2), the following experiments were conducted: 1. Cell Culture and Viral Infection: Normal human lung epithelial cells BEAS-2B and tumor cells A549, HELA and HepG2 in good growth condition were seeded into 6-well plates at an appropriate density and cultured overnight at 37°C and 5% CO2. When the cell confluence reaches 70%~80%, discard the original culture medium and gently wash twice with PBS; The experimental group was given PCV2 virus solution with an MOI of 1 (PCV2 virus obtained in Example 1 was diluted with serum-free medium), while the control group was given an equal volume of serum-free medium. After incubating at 37°C for 1.5 hours, the virus solution was discarded and replaced with maintenance medium containing 2% FBS. The culture was then continued for 24 hours for subsequent testing.

[0067] 2. PKM2 enzyme activity assay: Cell collection: Discard the culture medium, wash twice with pre-cooled PBS, add trypsin to digest and collect the cells; Preparation of lysis buffer: Add 200 μL of pre-chilled PKM2 enzyme activity assay lysis buffer (containing 1% protease inhibitor) to each well and lyse on ice for 30 minutes; Collect the supernatant: Centrifuge at 4℃ and 12000 rpm for 15 minutes, and collect the supernatant; Activity determination: The pyruvate kinase activity assay kit (Sigma-Aldrich) was used. The operation was strictly carried out in accordance with the instructions. The absorbance value was measured at a wavelength of 570 nm using an enzyme-linked immunosorbent assay (ELISA) reader. The PKM2 enzyme activity units were calculated based on the standard curve.

[0068] The results are as follows Figure 7 As shown, PCV2 infection significantly inhibited the enzyme activity of PKM2 in A549, Hela and HepG2 tumor cells (p<0.001), while having no significant effect on BEAS-2B normal cells (p>0.05).

[0069] 3. ATP content detection Cell treatment and grouping are the same as in step 1; Cell lysis: After culturing for 24 hours, discard the culture medium, wash twice with pre-cooled PBS, add 100 μL of ATP to each well to detect lysis buffer, and lyse on ice for 15 minutes; Centrifugation: Centrifuge at 4℃ and 12000 rpm for 5 minutes, and collect the supernatant; ATP concentration determination: The ATP assay kit (Beyotime) was used. The lysis buffer was mixed with the ATP assay working solution according to the instructions. The luminescence value was detected using a chemiluminescence analyzer, and the ATP concentration was calculated based on the standard curve.

[0070] The results are as follows Figure 8 As shown, PCV2 infection significantly reduced intracellular ATP levels in tumor cells (p<0.001), but did not affect normal BEAS-2B cells (p>0.05).

[0071] 4. Observation of culture medium acidification and detection of lactic acid 4.1 Observation of culture medium acidification The cell inoculation, virus infection, and culture methods are the same as in step 1; After 24 hours of incubation, the color change of the culture medium was directly observed and photographed. The results are as follows: Figure 9 As shown, the culture medium for tumor cells not infected with PCV2 turned significantly yellow (acidic), while the culture medium for infected cells remained red (neutral / alkaline).

[0072] 4.2 Lactic acid content detection Collect culture supernatant: After culturing for 24 hours, collect the cell supernatant from each group and centrifuge at 12,000 rpm for 5 minutes to remove cell debris; Lactic acid determination: The lactic acid assay kit (Sigma-Aldrich) was used. The absorbance value was measured at a wavelength of 450 nm according to the instructions. The lactic acid concentration was calculated based on the standard curve.

[0073] The results are as follows Figure 10As shown, PCV2 infection significantly inhibited lactate production in tumor cells (p<0.001), but had no effect on BEAS-2B cells (p>0.05).

[0074] The above experimental results indicate that PCV2 infection can specifically inhibit the activity of PKM2 enzyme in tumor cells, block glycolysis, reduce ATP production and lactate secretion, thereby altering the acidic tumor microenvironment. This effect is likely one of the key mechanisms by which PCV2 selectively targets tumor cells and exerts its oncolytic effect.

[0075] Example 5 PCV2 inhibits tumor cell migration and invasion. The inhibitory effect of PCV2 virus on the migration and invasion ability of A549 cells was verified through the following experimental steps: 1. Cell scratch assay: Cell culture: A549 cells were seeded in 6-well plates and cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2 until the confluence reached more than 90%. Scratch preparation: Use a 200μL sterile pipette tip to vertically scratch a uniform scratch on the cell monolayer in each well; PBS washing: Gently wash the cells three times with pre-warmed PBS buffer to remove detached cells; Drug treatment: The experimental group was given culture medium containing PCV2 virus (MOI=1), and the control group was given an equal volume of blank culture medium; Image recording: Images of the scratched area were acquired at 12h, 24h and 48h under an inverted microscope.

[0076] The results are as follows Figure 11 As shown in Figure A, compared with the uninfected control group, the cell scratch closure rate of the PCV2-infected group was significantly slowed down, indicating that PCV2 can effectively inhibit the migration ability of A549 cells.

[0077] 2. Transwell chamber cell invasion assay Pretreatment of the chamber: Matrigel coating in the upper chamber of Transwell (8 μm pore size, Corning): Dilute Matrigel (BD Biosciences) with serum-free medium at a ratio of 1:8, take 100 μL and spread it evenly on the membrane surface of the upper chamber, and cure at 37°C for 1 h; Cell seeding: Prepare cell suspension (5 × 10⁻⁶) 5 The experimental group was pretreated with PCV2 (MOI=1) for 24 h, while the control group received no treatment. 200 μL of cell suspension was added to the upper chamber, and 600 μL of DMEM medium containing 20% ​​FBS was added to the lower chamber. Culture and fixation: After culturing at 37℃ and 5% CO2 for 24 h, the chamber was removed and fixed with 4% paraformaldehyde for 30 min. Staining and counting: The cells were stained with 0.1% crystal violet for 20 min, gently rinsed with PBS, and the number of cells that had perforated the membrane was counted in 5 randomly selected fields of view under a microscope. The average value was then used for statistical analysis.

[0078] The results are as follows Figure 11 As shown in B, the number of transmembrane cells in the PCV2-infected group was significantly less than that in the control group, demonstrating that PCV2 also has a significant inhibitory effect on the invasive ability of A549 cells.

[0079] The above experimental results indicate that PCV2 infection can significantly inhibit the migration and invasion behavior of A549 cells.

[0080] Example 6 Validation of PCV2 oncolytic efficacy in A549 cell xenograft tumor BALB / C nude mouse model This experiment consisted of two groups, with each group using 6 BALB / c nude mice. Approximately 2 × 10⁻⁶ mg / L was injected subcutaneously into the same site in each mouse. 6 A549 cells. The tumor was induced to grow to an average size of approximately 100 cm². 3 Subsequently, the experimental group (PCV2 group) underwent intratumoral injection of 50 μL of PCV2 virus solution (titer of 5 × 10⁻⁶). 6 PFU / cell was administered to the control group (NC group or PBS group), while the control group was injected with an equal volume of placebo (PBS). Tumor volume was measured every 3 days starting from day 5 post-injection, and counted when the tumor volume exceeded 500 cm³. 3 Once the experimental phenomena become clear, the mice are euthanized, the subcutaneous tumor is completely dissected, weighed, and pathological HE staining, immunofluorescence, and immunohistochemical detection are performed to verify the histological characteristics of the tumor model.

[0081] The results are as follows Figure 12 As shown, in terms of tumor size, the tumor volume in the PCV2 treatment group was significantly smaller than that in the placebo group; in terms of morphology, the tumors in the placebo group exhibited multifocal growth, while those in the PCV2 group showed a single, mass-like structure; regarding the difficulty of anatomical dissection, the tumors in the placebo group often invaded surrounding muscle tissue, making dissection difficult, while the tumors in the PCV2 group had clear boundaries, no infiltration, and were easy to dissect. These results indicate that PCV2 treatment can significantly inhibit tumor growth and has the potential to inhibit tumor migration and invasion.

[0082] Depend on Figure 13 and Figure 14It can be seen that the tumor growth curve in the PCV2 treatment group was significantly flatter, and the final tumor weight was significantly lower than that in the placebo group, with a tumor inhibition efficiency of over 50%, further confirming that PCV2 has a significant inhibitory effect on tumor growth.

[0083] Subsequently, the infection status of PCV2 in the tumor tissue was identified, and the results were as follows: Figure 15 As shown, no positive signal of PCV2 was detected in the placebo group, while the virus was widely distributed in the vast majority of tumor cells in the PCV2 infection group, indicating that intratumoral injection of PCV2 can achieve efficient infection in vivo.

[0084] To further evaluate the oncolytic effect of PCV2 on solid tumors, both groups of tumor tissues were stained with hematoxylin and eosin (HE), and their overall morphology was visualized using panoramic scanning images. The results are as follows: Figure 16 As shown in the magnified view, in the placebo group, tumor cells were densely packed, with an increased nucleus-to-cytoplasmic ratio, deeply stained nuclei, active mitotic figures, less stroma, and intact structure, with no obvious necrosis or inflammatory response. Conversely, the PCV2-treated group showed extensive necrosis areas, characterized by pyknosis and fragmentation of cell nuclei, increased eosinophilicity of the cytoplasm, blurred cell boundaries, significant stroma edema, loose tissue structure, and scattered residual tumor cells. This demonstrates that PCV2 can effectively induce tumor cell death in vivo and exert an oncolytic effect.

[0085] To clarify the mechanism of PCV2-induced cell death, TUNEL staining was used to detect apoptosis. TUNEL staining is based on the principle of specific labeling of DNA breaks during apoptosis. Panoramic scan images are shown below. Figure 17 As shown in the magnified image, there was almost no apoptosis-related red fluorescence signal in the placebo group, while the PCV2 group showed a large number of red fluorescence positive signals in the necrotic areas of condensed and fragmented nuclei, indicating that PCV2 mainly mediates tumor cell death by inducing apoptosis.

[0086] Further observation through direct examination of tumor tissue sections yielded the following results: Figure 18 As shown, the tumors in the placebo group exhibited invasive behavior into the surrounding muscle tissue, and metastatic lesions were observed to form on the inner side of the muscle; while the tumors in the PCV2 infection group were encapsulated by subcutaneous connective tissue with clear boundaries, and no signs of invasion or metastasis were found, proving that PCV2 infection can inhibit the invasion and metastasis of tumors.

[0087] Throughout the experiment, the mice's mental state, feeding behavior, and weight changes were continuously monitored. Although a few animals in the PCV2 inoculation group experienced transient weight loss (the difference was not statistically significant, and data were not shown), no other adverse reactions were observed. Pathological examination of the major organs was performed after dissection, and the results are as follows: Figure 19As shown, no significant pathological changes were observed in the PCV2 infection group, nor were lesions such as dermatitis, kidney white spots, and lymph node enlargement commonly seen in porcine hosts, indicating that PCV2, as an oncolytic virus, has good safety in non-specific hosts.

[0088] Example 7 HeLa cell xenograft BALB / c nude mouse model to verify the oncolytic effect of PCV2. To evaluate the oncolytic effect of PCV2 on another human cancer, HeLa cells were used to construct a nude mouse xenograft tumor model. The experimental grouping and operation methods were the same as in Example 6.

[0089] like Figure 20 As shown, compared with the control group, the tumor volume formed by HeLa cells in the PCV2 treatment group was significantly reduced, indicating that PCV2 also has a significant inhibitory effect on the in vivo growth of HeLa cells.

[0090] From the tumor growth curve (see) Figure 21 As can be seen, from day 3 after PCV2 injection, the tumor growth rate in the experimental group was significantly lower than that in the placebo group. By day 33 of the experiment, the tumor inhibition rate in the PCV2-treated group reached approximately 80%. Furthermore, the tumor weight statistics collected at the endpoint (see...) Figure 22 Further results showed that PCV2 treatment significantly reduced tumor mass, consistent with volume measurements, fully demonstrating the strong inhibitory effect of PCV2 on the growth of HeLa cell xenografts.

[0091] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of PCV2 in the preparation of oncolytic viruses.

2. Application of PCV2 in the preparation of tumor treatment products.

3. The application according to claim 1 or 2, characterized in that, The nucleotide sequence of the PCV2 is shown in SEQ ID NO.

1.

4. The application according to claim 2, characterized in that, The tumors include lung cancer, cervical cancer, liver cancer, pancreatic cancer, breast cancer, lymphoma, or monocytic leukemia.

5. Application of PCV2 in the preparation of products that promote tumor cell apoptosis.

6. Application of PCV2 in the preparation of products that inhibit tumor cell migration.

7. Application of PCV2 in the preparation of products that inhibit tumor cell invasion.

8. The application according to any one of claims 5 to 7, characterized in that, The tumor cells include human lung cancer cells A549, human cervical cancer cells HeLa, human liver cancer cells HepG2, human pancreatic cancer cells PANC-1, human breast cancer cells MCF-7, lymphoma U937, or monocytic leukemia THP-1.

9. An oncolytic virus, characterized in that, Includes PCV2, wherein the effective dose of PCV2 is 1~10×10 6 TCID 50 / mL.

10. The use of the oncolytic virus of claim 9 in the preparation of tumor treatment products.

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