Application of fucoidin in preparation of preparation for resisting goose parvovirus infection

Abstract

The invention relates to an application of fucoidin (FUC) in preparation of a preparation for resisting goose parvovirus (GPV) infection, primary muscovy duck embryo small intestine epithelial cells (PMDIECs) and GPV PT isolates are used as research objects, and through the technologies of cell activity detection, immunoblotting, absolute fluorescence quantification qPCR, half tissue cell infection dose, immunofluorescence analysis and the like, the preparation of the preparation for resisting goose parvovirus (GPV) infection is prepared. The system carries out FUC cytotoxicity determination, in-vitro anti-GPV infection evaluation and action mechanism exploration. Results show that no obvious cytotoxicity exists when the FUC with the concentration of 6 mg / mL acts on PMDIECs for 72 h, the FUC can inhibit GPV infection in a dose-dependent mode, GPVVP3 protein expression in cells, the virus genome copy number, the culture supernatant virus titer and the like are remarkably reduced, it is confirmed for the first time that the FUC has the anti-GPV activity in vitro, and an experimental basis is provided for research and development of veterinary preparations for preventing and controlling GPV infection.

Description

Technical Field

[0001] This invention belongs to the field of veterinary microbiology and antiviral technology of bioactive substances, specifically involving the application of fucoidan (FUC) in the preparation of anti-goose parvovirus (GPV) infection preparations, and related technologies for evaluating the in vitro inhibitory effect of fucoidan on goose parvovirus replication. Background Technology

[0002] Derzsy's disease is a highly contagious and devastating viral disease of waterfowl, primarily affecting geese and Muscovy ducks. The pathogen is goose parvovirus (GPV), belonging to the genus *Derzsy* 1 of the order Anseriformes in the family Parvoviridae. The virus particle has a non-enveloped icosahedral structure, composed of viral structural proteins, encapsulating a linear single-stranded DNA genome with inverted terminal repeats (ITRs) at both ends. This hairpin-dependent, parvovirus-specific rolling circle replication mechanism accelerates viral recombination and evolution. The GPV genome is approximately 5100 bp long, encoding non-structural proteins (NS1 and NS2) and viral capsid proteins (VP1, VP2, and VP3), which are expressed in large quantities through differential splicing. The NS proteins specifically bind to ITRs to initiate viral DNA replication and capsid gene expression. Simultaneously, structural proteins VP1, VP2, and VP3 form the viral capsid in a 1:1:8 ratio, creating an icosahedral structure, with VP3 being the primary capsid component. GPV primarily infects goslings and young Muscovy ducks under one month old, exhibiting typical clinical symptoms such as weight loss, conjunctivitis, and watery diarrhea. Furthermore, this infection often leads to acute enteritis, inflammatory responses in vital organs (such as the heart, liver, and kidneys), necrotizing enteritis, and intestinal embolism formed by coagulated fibrinous exudate, accompanied by necrosis and sloughing of the intestinal mucosa. GPV is mainly transmitted through the secretions and excretions of infected individuals, characterized by rapid spread and high mortality. Notably, goslings and young Muscovy ducks typically show no obvious symptoms after infection, but vertical transmission to offspring can occur, allowing GPV to form an intergenerational cycle through hatching eggs, leading to embryonic death, reduced hatchability, and the covert spread of GPV within the breeding population. This exacerbates the risk of secondary infections and further increases the difficulty of clinical control of gosling plague.

[0003] Fujian Province is not only an important base for the Muscovy duck industry but also a high-incidence area for GPV infection. Currently, prevention and control of Muscovy duck gosling plague mainly relies on vaccination. However, inactivated vaccines are difficult to produce sufficient neutralizing antibodies, and attenuated vaccines cannot produce complete immunity against multiple viral strains. Furthermore, the lack of effective antiviral drugs further increases the difficulty of comprehensive prevention and control. Fucoidan (FUC) is a sulfated polysaccharide mainly extracted from brown algae. Its complex structure includes L-fucose, sulfate ester groups, and monosaccharides (such as galactose, mannose, and xylose), which endow it with diverse biological activities. In existing technologies, fucoidan has not yet been confirmed to possess broad-spectrum antiviral activity. Current research on the antiviral mechanism of fucoidan targets specific enveloped virus groups and has not yet explored the field of non-enveloped viruses. The enveloped virus group includes herpesviruses (enveloped DNA viruses), a type of double-stranded DNA virus, and coronaviruses (enveloped RNA viruses), a type of single-stranded RNA virus (e.g., prior art CN 115835870 A discloses an antiviral drug composition including fucoidan, which targets coronaviruses). Goose parvovirus (GPV) is a typical non-enveloped single-stranded DNA virus, and its viral particle structure (no lipid envelope) and genome type (single-stranded linear DNA) are fundamentally different from those of previously studied enveloped viruses. A search of domestic and international literature (including PubMed, CNKI, Web of Science, etc.) revealed no reports of the inhibitory effect of fucoidan on non-enveloped single-stranded DNA viruses. Therefore, regarding whether fucoidan (FUC) has a biological function in inhibiting GPV infection, no relevant reports have been found in the currently searched literature. In this study, the antiviral effect of fucoidan on GPV infection was investigated, and its antiviral mechanism was further explored. According to the data presented in this invention, FUC exhibits significant antiviral effects in the early stages of GPV infection, effectively inhibiting its activity by interfering with multiple stages of the GPV life cycle (including viral attachment, internalization, replication, and release). The results of this invention suggest that FUC has potential for veterinary clinical application in the treatment of GPV infection. This invention establishes for the first time the antiviral association between this type of virus and fucoidan, directly filling the gap in fucoidan antiviral research in the field of "non-enveloped single-stranded DNA viruses." Summary of the Invention

[0004] The purpose of this invention is to address the technical problem of limited GPV infection prevention and control methods in existing technologies. Therefore, this invention provides an application of FUC in the preparation of anti-GPV infection agents, aiming to provide a new technical solution and material selection for GPV infection prevention and control.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] Application of fucoidan (FUC) in the preparation of agents against goose parvovirus (GPV) infection.

[0007] The anti-goose parvovirus (GPV) infection preparation includes preparations that inhibit goose parvovirus (GPV) adsorption to host cells and / or inhibit goose parvovirus (GPV) infection of host cells.

[0008] The anti-goose parvovirus (GPV) infection preparations include preparations for the prevention and / or treatment of goose parvovirus (GPV) infection.

[0009] The anti-goose parvovirus (GPV) infection preparation includes preparations that interfere with the life cycle of goose parvovirus (GPV).

[0010] The life cycle includes viral adsorption, cell entry, replication, and release.

[0011] The preparation is a veterinary drug preparation.

[0012] The dosage forms of the veterinary drug preparations include powders, oral liquids, and soluble powders.

[0013] The goose parvovirus strain mentioned is GPV-PT. In evaluating the effects of FUC on GPV infection activity, viral life cycle interference, viral adsorption, viral entry inhibition, and viral replication and release, the goose parvovirus isolate used in this invention is GPV-PT.

[0014] The application concentration of the fucoidan (FUC) is 0.75-6 mg / mL.

[0015] Compared with the prior art, the advantages of the present invention are as follows:

[0016] (1) FUC is derived from abundant brown algae resources, with mature extraction technology, easy to scale up production, and controllable cost.

[0017] (2) FUC showed significant anti-GPV infection effects, effectively inhibiting the adsorption and invasion of GPV into primary Muscovy duck embryonic intestinal epithelial cells (PMDIECs), and significantly reducing viral proliferation titers.

[0018] (3) FUC is a natural marine bioactive substance with good biocompatibility and high toxicological safety, and is suitable for long-term prevention and control of sensitive groups such as ducklings.

[0019] (4) FUC has multiple biological functions such as antiviral, immune enhancement and intestinal barrier protection. In view of the shortcomings of inactivated vaccines (such as relatively slow immune response and often requiring multiple vaccinations to maintain effective immune levels) and attenuated vaccines (such as potential risk of virulence reversion and poor safety for immunosuppressed individuals), it has a synergistic advantage in the comprehensive prevention and control of GPV infection. Attached Figure Description

[0020] Figure 1 The results of the toxic effects of FUC on PMDIECs in Example 3 of this invention;

[0021] Figure 2 The results of FUC inhibiting GPV infection activity on PMDIECs in Example 4 of this invention are shown, where A, B, C, and D represent Western blotting, qPCR, and TCID, respectively. 50 The effect of FUC on GPV infection on PMDIECs was determined using IFA (scale bar = 100 m);

[0022] Figure 3 The results of FUC inhibiting GPV infection of PMDIECs in Example 5 of this invention under three administration methods [pre-treatment, co-treatment, and post-treatment] are shown, where A and B are qPCR and TCID, respectively. 50 The effect of FUC on GPV infection in PMDIECs was determined; C, D, and E were the effects of FUC on GPV VP3 protein expression in PMDIECs under three administration methods determined by Western blotting.

[0023] Figure 4 The effect of FUC on GPV adsorption of PMDIECs in Example 6 of this invention is shown in A and B, respectively, for qPCR and Western blotting results.

[0024] Figure 5 The effect of FUC on GPV entry into PMDIECs in Example 7 of this invention is shown in A and B, respectively, for qPCR and Western blotting results.

[0025] Figure 6 This is the effect of FUC on GPV proliferation in PMDIECs in Example 8 of the present invention. In Example 8 of the present invention, A is the effect of FUC on the copy number of GPVVP3 gene at 12 hpi and 24 hpi, and B and C are the effects of FUC on the expression level of GPVVP3 protein at 12 hpi and 24 hpi, respectively.

[0026] Figure 7 This illustrates the effect of FUC on GPV release in Example 9 of the present invention. A and B represent qPCR and TCID, respectively. 50 result;

[0027] Figure 8 To illustrate the effect of FUC on the relative expression levels of pro-inflammatory factors and type I interferon mRNA induced by GPV in PMDIECs in Example 10 of the invention, GPV-infected PMDIECs were divided into two groups and incubated at 37°C with or without 6 mg / mFUC. Total RNA was extracted from cell lysates at 12, 24, 36, 48, 60, and 72 h after treatment. Interleukin-1β (IL-1β) was detected using RT-qPCR. Figure 8 A) Interleukin-6 (IL-6, Figure 8 B) Interleukin-8 (IL-8, Figure 8 C) Tumor necrosis factor-α (TNF-α) Figure 8 D) Interferon-α (IFN-α) Figure 8 E) and interferon-β (IFN-β, Figure 8 The relative mRNA expression level of F). This experiment was performed three independent replicates under the same conditions. The results are expressed as mean ± standard deviation. Statistical analysis was performed with the GPV-infected group as the control. The significance level is marked as follows: * represents p<0.05, ** represents p<0.01, and *** represents p<0.001. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0029] It should be noted that, unless otherwise defined, the technical or scientific terms used in the embodiments of the present invention should have the ordinary meaning as understood by one of ordinary skill in the art to which this disclosure pertains.

[0030] Example 1

[0031] Preparation of cells and viruses

[0032] Primary Muscovy duck embryonic intestinal epithelial cells (PMDIECs) were isolated from the small intestine tissue of healthy 27-day-old Muscovy duck embryos. Cell preparation and culture followed standard experimental procedures. During the culture phase, Dulbecco's Modified Eagle Medium (DMEM) complete medium containing 10% fetal bovine serum was used to ensure normal cell growth and viability. The GPV-PT virus strain used in this study was isolated and preserved in our laboratory.

[0033] Example 2

[0034] Preparation of FUC formulations

[0035] Fucoidan (FUC, Shanghai Aladdin Biochemical Technology Co., Ltd.) was dissolved in DMEM culture medium to prepare a fucoidan preparation with a storage concentration of 24 mg / mL. The preparation was sterilized by filtration through a 0.22 μm filter membrane, appropriately aliquoted, and stored at -20℃.

[0036] Example 3: Determination of the toxicity of FUC to PMDIECs

[0037] First, PMDIECs are divided into 1×10 5 FUCs were seeded at a concentration of [number] cells / mL into 96-well plates. A blank control group was also set up using maintenance medium (DMEM containing 2% fetal bovine serum) without any cells. FUCs were serially diluted two-fold with maintenance medium and co-cultured with PMDIECs in 96-well plates for 72 h. A negative control group containing only maintenance medium was also set up. After 72 h, 10 μL of CCK-8 reagent (Shanghai Beyotime Biotechnology Co., Ltd.) was added to each well, and the plates were incubated at 37°C in the dark for 2 h. The concentration of FUCs at 450 μL was measured using a microplate reader. nm Absorbance values. The results showed that treatment of PMDIECs with 6 mg / mL FUC for 72 h did not exhibit significant cytotoxicity. Figure 1 ).

[0038] Example 4: The inhibitory activity of FUC on GPV infection in PMDIECs

[0039] (1) Western blotting to determine the inhibitory activity of FUC on PMDIECs against GPV infection

[0040] PMDIECs were cultured in 24-well plates at 37°C to 80% confluence. The complete culture medium was discarded, and the cells were washed three times with PBS. Subsequently, the cells were pretreated in maintenance culture medium with FUC (0, 0.75, 1.5, 3, and 6 mg / mL) for 2 h. The cell culture supernatant was discarded, and the cells were washed three times with PBS. The cells were then infected with GPV PT virus strain with a multiplicity of infection (MOI) of 1 at 37°C for another 2 h. Afterward, the virus solution was removed, and the plates were washed three times again with PBS. Maintenance culture medium containing different concentrations of FUC (final concentration same as the pretreatment stage) was added to the cells, and incubation continued for 70 h. A control group containing only maintenance culture medium and no FUC was also established. 72 hours after virus infection, cell culture supernatant and cell samples were collected. The cells were washed three times with PBS, and the viral VP3 protein level in GPV-infected cells was analyzed by Western blotting to determine the antiviral activity of FUC. The results showed that exogenous addition of FUC significantly reduced the expression level of GPVVP3 protein in PMDIECs-infected cells, preliminarily confirming that FUC has the potential to interfere with the infection process of GPV in host cells and inhibit its infectivity. Figure 2 A).

[0041] (2) Determination of GPVVP3 gene copy number in PMDIECs by quantitative real-time polymerase chain reaction (qPCR)

[0042] Total DNA extraction from cell samples was performed according to the instructions of the FastPure Cell / Tissue DNA Isolation Mini Kit (Nanjing Novizan Biotechnology Co., Ltd.). Primers for the GPVVP3 gene were designed based on the previously established GPV SYBR Green I qPCR detection method in our laboratory and synthesized by Fuzhou Shangya Biotechnology Co., Ltd., with sequences of GPV G5P1F (5'-GAGGTAGACAGCAACAGAAA-3') and GPV G5P2R (5'-GCTCGTCCGTGACCATA-3'). qPCR analysis was performed using the ChamQ Blue Universal SYBR qPCR MasterMix (Nanjing Novizan Biotechnology Co., Ltd.), with the reaction program set as follows: 95℃ denaturation for 30 seconds, followed by 40 cycles of 95℃ denaturation for 10 seconds and 60℃ annealing for 30 seconds. A standard curve was plotted using the pre-constructed pMD19-T-GPV plasmid (pGDA) as the standard plasmid. The GPV copy number was calculated from this curve to detect the GPV viral load in the infected samples. The results showed that FUC significantly inhibited the replication and proliferation of GPV in infected PMDIECs, and this inhibitory effect was concentration-dependent. Figure 2 B).

[0043] (3)TCID 50 Determining the inhibitory activity of FUC on PMDIECs against GPV infection.

[0044] Cell culture supernatants were collected from the GPV-infected control group and the FUC anti-GPV treatment groups at various concentrations (0, 0.75, 1.5, 3, and 6 mg / mL) in Example 4(1). The cell culture supernatants collected in Example 4(1) were serially diluted 10-fold. They were then seeded into 96-well plates containing a monolayer of PMDIECs with 80% cell confluence, with 8 wells per dilution and 100 μL per well. After 2 hours of adsorption, the cells were washed 2-3 times with PBS buffer, and then an equal volume of cell maintenance medium was added. The cells were cultured continuously at 37°C for 7 days. A normal cell control group was also included. Cytopathic effects were observed and recorded daily. Wells with ≥50% cell cytopathic effects were considered positive wells. The number of positive wells at each dilution was recorded, and TCID was calculated using the Reed-Muench method. 50 The results showed that FUC could reduce the viral titer in cell culture supernatant in a dose-dependent manner. Figure 2 C) demonstrates that FUC has a significant inhibitory effect on the infection process of GPV.

[0045] (4) Immunofluorescence assay (IFA) was used to determine the inhibitory activity of FUC on PMDIECs against GPV infection.

[0046] PMDIECs were treated according to the method in Example 4(1). After 72 h of infection, the cells were washed three times with PBS, covered with 4% paraformaldehyde pre-cooled at 4°C, fixed at 37°C for 15 min, and then the fixative was discarded. The cells were washed three times with PBS for 5 min each time. 0.1% Triton X-100 permeabilizer was added, and the cells were incubated at room temperature for 5-10 min. The permeabilizer was discarded, and the cells were washed three times with PBS for 5 min each time. 5% BSA blocking solution was added to the culture wells to cover the cell surface, and the cells were blocked overnight at 4°C. After blocking, anti-GPV monoclonal antibody (1:200) was added and the cells were incubated at 37°C for 1-2 h. After incubation, the primary antibody solution was discarded, and the cells were washed 3-4 times with PBS for 5-10 min each time. FITC-labeled goat anti-mouse IgG antibody (1:200) was added to the culture wells and the cells were incubated at room temperature in the dark for 30 min-1 h. After incubation, the secondary antibody solution was discarded, and the cells were washed 3-4 times with PBS for 5-10 minutes each time, protected from light. DAPI staining solution was added to the culture wells, and the cells were incubated at room temperature in the dark for 5-10 minutes before observation. The results showed that FUC could reduce the viral titer in the cell culture supernatant in a dose-dependent manner. Figure 2 D) demonstrates that FUC can inhibit the infection and proliferation of GPV in PMDIECs.

[0047] Example 5: Inhibitory effect of FUC on GPV infection under different administration methods

[0048] PMDIECs were cultured in 24-well plates at 37°C with 5% CO2 until confluence was achieved. Subsequently, during GPV infection, they were administered FUC (0, 0.75, 1.5, 3, and 6 mg / mL) via three routes: pre-treatment, co-treatment, and post-treatment. The specific procedures are briefly described below:

[0049] In the prophylactic administration group, PMDIECs were first incubated with FUC at 37°C for 2 hours at concentrations of 0, 0.75, 1.5, 3, and 6 mg / mL, respectively. The FUC-containing cell maintenance culture medium was then discarded. After three washes with PBS, cells were infected with the GPV PT strain (MOI=1) for 2 hours. The virus solution was then discarded, and the cell monolayers were washed three times with PBS, followed by replenishment with fresh FUC-free cell maintenance culture medium. A blank cell control group containing only maintenance culture medium and no FUC was also established.

[0050] In the concurrent drug administration group, an equal volume of maintenance medium containing FUC (0, 1.5, 3, 6, and 12 mg / mL) was mixed with GPV PT strain (MOI=2), and co-incubated at 37°C for 2 hours. The mixture was then inoculated into PMDIECs and cultured for another 2 hours. The GPV and FUC mixture was discarded, and after three washes with PBS, fresh maintenance medium without FUC was added and the cells were cultured again at 37°C. A control group containing only maintenance medium and no FUC was also established.

[0051] In the treatment group, PMDIECs were first incubated with GPV (MOI=1) at 37°C for 2 hours. The viral load was then discarded, and the cells were washed three times with PBS. Fresh cell maintenance culture medium containing FUC (concentrations of 0, 0.75, 1.5, 3, and 6 mg / mL) was then added, and incubation continued at 37°C. A control group containing only maintenance culture medium and no FUC was also established.

[0052] Throughout the entire experiment of Example 5, all cells treated as described above were cultured for 72 hours post-infection, at which point infected cells and their culture supernatant were collected. The GPV genome copy number was determined by absolute quantitative qPCR using TCID50. 50 Viral titer was detected by a specific method, and the expression level of GPV VP3 protein was analyzed by Western blotting. The results showed that FUC could inhibit the infectivity of GPV against PMDIECs under three different administration methods. In particular, the direct inactivation effect of FUC on GPV was significantly significant under the simultaneous administration mode, significantly inhibiting GPV VP3 protein expression, reducing viral copy number, and decreasing the viral titer in the supernatant of infected cells. Figure 3 AE).

[0053] Example 6: Effect of FUC on GPV attachment

[0054] PMDIECs that had grown into a monolayer in 24-well cell culture plates were pre-cooled at 4°C for 30 min, then inoculated with GPV-PT (MOI = 1) and cell maintenance medium containing different concentrations of FUC (0, 0.75, 1.5, 3, 6 mg / mL), and incubated together at 4°C for 2 h. A blank control group was also set up, incubating PMDIECs only with cell maintenance medium without FUC. After incubation, the supernatant was discarded, and the cells were washed three times with pre-cooled PBS. Cell samples were then collected, and the GPV genome copy number was determined by absolute quantitative qPCR, and the expression level of GPV-VP3 protein was detected by Western blotting. The qPCR and Western blotting results showed that treatment with FUC significantly reduced the amount of GPV adsorbed on the surface of PMDIECs, inhibiting VP3 protein expression, demonstrating that FUC can inhibit the adsorption of GPV to PMDIECs. Figure 4 A, B).

[0055] Example 7: Effect of FUC on GPV Entry

[0056] PMDIECs were seeded into 24-well cell culture plates. After reaching 90% confluence, the cells were pre-cooled at 4°C for 30 min and then infected with GPV-PT (MOI=1) for 2 h at the same temperature. The cells were then washed three times with cold PBS to remove unbound viral particles. Subsequently, the infected cells were treated with maintenance medium containing 0, 0.75, 1.5, 3, and 6 mg / mL FUC for 2 h at 37°C to promote viral entry. A blank control group was maintained in the same maintenance medium without viral infection. After incubation, the cells were washed three times with a highly alkaline, high-salt solution [1 M sodium chloride and 50 mM tris(hydroxymethyl)aminomethane (pH 9.5)] to remove uninternated extracellular viruses. Cells were then collected for absolute quantitative qPCR analysis of viral DNA copy number, and GPV-VP3 protein expression level was analyzed by Western blotting. The qPCR and Western blotting results showed that FUC could inhibit GPV entry into PMDIECs (… Figure 5 A, B), and the inhibitory effect of FUC on the GPV entry process is more significant than its effect on the GPV adsorption of PMDIECs.

[0057] Example 8: The effect of FUC on GPV replication

[0058] PMDIECs were seeded into 24-well cell culture plates. After cells reached 90% confluence, they were inoculated with GPV-PT (MOI = 1) at 37°C for 4 hours. After infection, maintenance culture medium containing 0, 0.75, 1.5, 3, and 6 mg / mL FUC was added to each group, respectively. A blank control group (containing only FUC-free maintenance culture medium) was also set up. All groups continued to be incubated at 37°C. Cell samples were then collected at 12 hours post-infection (hpi) and 24 hours post-infection. Absolute quantitative PCR (qPCR) and Western blotting were used to detect the copy number of the GPVVP3 gene and its protein expression level. The qPCR and Western blotting results showed that FUC effectively inhibited the replication and proliferation of GPV in PMDIECs. Figure 6 AC).

[0059] Example 9: The effect of FUC on GPV release

[0060] PMDIECs were seeded into 24-well cell culture plates. After the cells reached 90% confluence, they were inoculated with GPV-PT (MOI=1) at 37°C for 24 h. The cells were then washed three times with PBS, and maintenance culture medium containing 0, 0.75, 1.5, 3, and 6 mg / mL FUC was added to the cells, respectively. The cells were incubated at 37°C for another 8 h. A blank control group (containing only maintenance culture medium without FUC) was also included. After incubation, cell samples and culture supernatants were collected from each group. Absolute quantitative qPCR was used to determine the GPV DNA copy number in the cells, and TCID50 was used to detect the viral titer in the cell culture supernatant. qPCR and Western blotting results showed that FUC effectively inhibited the release of GPV progeny virus. Figure 7 A, B).

[0061] Example 10: Effects of FUC on the expression of inflammatory factors and type I interferon mRNA in GPV-infected PMDIECs

[0062] PMDIECs were treated according to the method in Example 4(1). After 72 hours of infection, total RNA was extracted from PMDIECs using the FastPure Cell / Tissue Total RNA Isolation KitV2 (Nanjing Novizan Biotechnology Co., Ltd.), and cDNA was synthesized using the HiScript IIQ Select RT SuperMix for qPCR (+gDNAwiper) (Nanjing Novizan Biotechnology Co., Ltd.) according to the manufacturer's instructions. The relative expression levels of interleukin-1β (IL-1β), IL-6, IL-8, tumor necrosis factor-α (TNF-α), interferon-α (IFN-α), and IFN-β mRNA in PMDIECs were detected using the ChamQ Blue Universal SYBR qPCRMasterMix (Nanjing Novizan Biotechnology Co., Ltd.). Primer sequences are shown in Table 1 and were synthesized by Fuzhou Shangya Biotechnology Co., Ltd. Reaction system: ChamQ Universal SYBRqPCR Master Mix 5.0 μL, sterile enzyme-free water 3.6 μL, template cDNA 1.0 μL, forward and reverse primers (10 pmol / μL) 0.2 μL each. Reaction program: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 10 s, 60℃ annealing and extension for 30 s, for a total of 40 cycles. β-Tubulin was used as an internal control. -ΔΔCt The relative mRNA expression levels of IL-1β, IL-6, IL-8, TNF-α, IFN-α, and IFN-β genes were calculated. Results showed that FUC significantly reduced the levels of pro-inflammatory cytokines IL-1β, IL-6, IL-8, and TNF-α in GPV-infected cells, with higher doses showing better results. Simultaneously, the expression levels of type I interferons (IFN-α and IFN-β) were significantly increased in the FUC-treated groups. Figure 8 AF).

[0063] Table 1 Primer sequence information for quantitative RT-qPCR

[0064]

[0065] As can be seen from the above embodiments:

[0066] This invention provides the first in vitro experimental demonstration that FUC has a significant inhibitory effect on GPV infection. The GPV life cycle can be divided into four stages: 1) viral particles attaching to the cell surface; 2) viral particles being internalized by the host cell; 3) viral particles replicating their genome; and 4) viral particles being released. Each stage may be a potential target for antiviral drug development. Through drug administration time experiments, this invention found that as the drug concentration increases, the viral titer gradually decreases in a dose-dependent manner, and the relative expression level of the GPVVP3 gene also gradually decreases accordingly. These differences are highly significant compared to the viral control group. At the same drug concentration, comparisons of different administration methods show that FUC has a significant direct inactivation effect on GPV.

[0067] The initial stage of viral infection involves the binding of the virus to cell surface receptors, followed by a stable and irreversible adsorption process that initiates subsequent cell invasion. Compared to post-infection antiviral drugs, inhibiting viral adsorption to the cell surface is a more effective strategy for preventing infection. At 4°C, the virus can only adsorb onto the cell membrane surface and cannot penetrate into the cytoplasm; this characteristic makes 4°C an ideal experimental condition for evaluating the specific effects of viral adsorption. Based on this advantage, this invention further evaluated the regulatory effect of FUC on the GPV adsorption process by treating Muscovy duck embryonic small intestinal epithelial cells with FUC. The results showed that compared with the untreated control group, the adsorption of GPV by FUC-treated cells was significantly reduced. Furthermore, even if the virus had initially adsorbed onto the cell surface, FUC could still block the internalization process of GPV. We hypothesize that the negative charge of the sulfate group in the FUC structure may interact with the positively charged groups on the viral capsid protein, thereby preventing the virus from binding to the cell, blocking adsorption, and interfering with replication; on the other hand, due to the gel properties of FUC, it may compete with the virus for binding to cell surface receptors, thereby inhibiting viral attachment. In antiviral experiments, FUC treatment effectively inhibited GPV replication. Notably, this inhibitory effect was more significant when targeting GPV replication than when targeting viral binding, internalization, or release. These findings suggest that interfering with viral protein synthesis is a key pathway for FUC to exert its antiviral activity.

[0068] GPV infection in goslings and young Muscovy ducks induces intestinal inflammation, primarily producing pro-inflammatory cytokines. These cytokines play a crucial role in the development of viral enteropathy in waterfowl by coordinating and activating adaptive immune responses, thereby helping the host clear the pathogen. Previous studies have shown that GPV can evade immune surveillance by host immune cells by downregulating type I interferon-1 (IFN-1)-dependent immune responses. Notably, the inventors of this invention found that GPV infection of susceptible host cells significantly increased the expression levels of IL-1β, IL-6, and TNF-α, consistent with previous findings. Furthermore, FUC treatment significantly reduced virus-induced cytokine expression and promoted the expression and transcription of IFN-α and IFN-β genes, thereby inhibiting GPV infection and replication in Muscovy duck embryonic small intestinal epithelial cells. These data indicate that FUC not only acts as an inhibitor of the viral life cycle but also modulates host inflammation and immune responses.

[0069] In summary, this invention is the first to demonstrate that FUC possesses significant antiviral activity against GPV. FUC exerts its effects in the early stages of infection by targeting key stages of the GPV life cycle, including viral adsorption, internalization, replication, and release, and can directly interact with GPV. This invention not only establishes the potential of FUC as a therapeutic agent for GPV infection but also provides a theoretical basis for its application in the prevention and treatment of GPV infection, and points the way for the future development of anti-GPV drugs.

[0070] Finally, it should be noted that those skilled in the art should understand that the discussion of any of the foregoing embodiments is merely exemplary and is not intended to imply that the scope of protection of this disclosure (including the scope defined by the claims) is limited to such examples. Within the technical concept framework of this invention, the technical features contained in the foregoing embodiments or different embodiments can be further combined with each other, and the execution order of related steps is also arbitrary. Furthermore, this invention has many other modifications as described above in different technical dimensions. To ensure brevity, these modifications have not been described in detail.

[0071] The embodiments of this invention are intended to cover all alternatives, modifications, and variations that fall within the broad scope of protection defined by the appended claims. Therefore, any omissions, modifications, equivalent substitutions, and improvements made within the framework of the technical spirit and core principles of this invention should be included within the scope of protection of this invention.

Claims

1. Application of fucoidan in the preparation of agents against goose parvovirus infection.

2. The application according to claim 1, characterized in that: The anti-goose parvovirus infection preparation includes preparations that inhibit goose parvovirus adsorption to host cells and / or inhibit goose parvovirus infection of host cells.

3. The application according to claim 1, characterized in that: The anti-goose parvovirus infection preparations include preparations for the prevention and / or treatment of goose parvovirus infection.

4. The application according to claim 1, characterized in that: The anti-goose parvovirus infection preparation includes agents that interfere with the life cycle of goose parvovirus.

5. The application according to claim 4, characterized in that: The life cycle includes viral adsorption, cell entry, replication, and release.

6. The application according to any one of claims 1-4, characterized in that: The preparation is a veterinary drug preparation.

7. The application according to claim 6, characterized in that: The dosage forms of the veterinary drug preparations include powders, oral liquids, and soluble powders.

8. The application according to any one of claims 1-4, characterized in that: The strain of goose parvovirus mentioned is GPV-PT.

9. The application according to claim 1, characterized in that: The application concentration of the fucoidan is 0.75-6 mg / mL.

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