Use of the parapoxvirus-derived peptide Hepvirusin in the preparation of a composition for the treatment of water-borne viral diseases

Abstract

The application discloses application of antherin antitoxic peptide Hepvirusin in preparation of an aquatic virus-resistant composition. The antherin antitoxic peptide Hepvirusin consists of 21 amino acids and has a molecular weight of 2230.82 Dalton. The antherin antitoxic peptide contains three positively charged amino acid residues, and the isoelectric point of the antitoxic peptide is 8.53 according to the charge of the amino acid residues, so the antherin antitoxic peptide is a positively charged cationic polypeptide. The antherin antitoxic peptide has a significant antiviral effect on eel herpes virus, and can be used for preparing the aquatic virus-resistant composition and aquatic feed additive. The antherin antitoxic peptide has no cytotoxic effect on eel ovary cells (EO), eel kidney cells (EK) and eel spleen cells (ES).

Description

Technical Field

[0001] This invention belongs to the field of marine molecular biology technology, specifically relating to the application of the sea bass antiviral peptide Hepvirusin in the preparation of anti-aquatic virus compositions. Background Technology

[0002] Eels, generally found in brackish waters, are highly nutritious and a key industry in Fujian province. Fujian ranks first in China in eel production, variety, roasted eel output, eel feed production, and foreign exchange earnings from eel exports. However, in recent years, eel populations have declined sharply globally. Besides overfishing and water pollution, pathogen infection (mainly viruses, bacteria, or parasites) is a significant contributing factor. Eel "mucus septicemia" is widespread in Fujian province during seasons with water temperatures of 24-32℃. In the early stages, the mucus on the eel's body surface becomes noticeably loose and proliferates, eventually shedding. As the disease progresses, ulcers develop in the dermis; autopsy reveals gill rot, intestinal inflammation, and liver enlargement. Currently, artificial breeding of eels is not yet possible; juveniles are harvested from estuaries for farming, resulting in high costs and significant economic losses from disease outbreaks. Secondly, reports indicate that the probability of eel contracting this disease during eel farming is almost 100%, and sometimes multiple infections can occur, with a cumulative mortality rate of 30%-40%. Furthermore, eels infected with this disease are more susceptible to further bacterial or parasitic infections, leading to an increased mortality rate.

[0003] Viral infection control typically relies on vaccination or antiviral drugs. However, most vaccines are administered intraperitoneally, making large-scale implementation in aquaculture farms difficult. Existing nucleoside antiviral drugs can lead to drug resistance and toxic side effects. In recent years, numerous antiviral peptides have been discovered in animals, plants, and microorganisms. Antiviral peptides exert their effects extracellularly primarily through direct viral inactivation, binding to viral components, and targeting cellular receptors. Intracellularly, they work by inhibiting viral gene expression and regulating immunity and programmed cell death. Compared to traditional nucleoside antiviral drugs, peptides offer advantages such as high activity, low toxicity, and a lower likelihood of inducing drug resistance. While some peptides targeting marine animal viruses have been reported, research on eel herpesviruses is currently lacking. Summary of the Invention

[0004] The purpose of this invention is to provide the application of the sea bass antiviral peptide Hepvirusin in the preparation of anti-aquatic virus compositions.

[0005] Another object of the present invention is to provide an anti-aquatic virus composition.

[0006] Another object of the present invention is to provide the application of the sea bass antiviral peptide Hepvirusin in the preparation of aquatic feed additives.

[0007] Another object of the present invention is to provide an aquatic feed additive.

[0008] The technical solution of the present invention is as follows:

[0009] Application of Hepvirusin, an antiviral peptide for sea bass, in the preparation of antiviral compositions for aquatic organisms.

[0010] In a preferred embodiment of the present invention, the anti-aquatic virus composition has an inhibitory and killing effect on eel herpesvirus.

[0011] An anti-aquatic virus composition comprising the sea bass antiviral peptide Hepvirusin as its active ingredient.

[0012] In a preferred embodiment of the present invention, the active ingredient is the sea bass antiviral peptide Hepvirusin.

[0013] Application of Hepvirusin, an antiviral peptide for sea bass, in the preparation of aquatic feed additives.

[0014] In a preferred embodiment of the present invention, the aquatic feed additive has an inhibitory and killing effect on eel herpesvirus.

[0015] An aquatic feed additive whose active ingredient includes the sea bass antiviral peptide Hepvirusin.

[0016] In a preferred embodiment of the present invention, the active ingredient is the sea bass antitoxin peptide Hepvimsin.

[0017] The beneficial effects of this invention are:

[0018] 1. The sea bass antiviral peptide Hepvirusin of the present invention is composed of 21 amino acids with a molecular weight of 2230.82 Daltons, including 3 positively charged amino acid residues. Based on the charge of the amino acid residues, the isoelectric point of the antiviral peptide is predicted to be 8.53, which is a positively charged cationic polypeptide.

[0019] 2. The sea bass antiviral peptide Hepvirusin of the present invention has a significant antiviral effect on eel herpesvirus. It can be developed into an anti-aquatic virus composition and can also be used as an aquatic feed additive.

[0020] 3. The sea bass antiviral peptide Hepvirusin of the present invention does not have cytotoxic effects on eel ovary cells (EO), eel kidney cells (EK) and eel spleen cells (ES). Attached Figure Description

[0021] Figure 1The graph for detecting the cytotoxicity of the sea bass antiviral peptide Hepvirusin by MTS-PMS in Example 2 of this invention includes eel ovary cells (EO), eel kidney cells (EK), and eel spleen cells (ES). The horizontal axis represents the concentration of Hepvirusin (μM), and the vertical axis represents the cell proliferation rate (%).

[0022] Figure 2 The graph shows the antiviral activity of the sea bass antiviral peptide Hepvirusin against eel herpesvirus in Example 3 of this invention: the horizontal axis represents the concentration of Hepvirusin (μM), and the vertical axis represents the viral DNA accumulation rate (%).

[0023] Figure 3 The following is a one-step growth curve of the antiviral activity of the sea bass antiviral peptide Hepvirusin in Example 4 of the present invention: the horizontal axis is time (hour), and the vertical axis is the number of virus copies (copies / ng).

[0024] Figure 4 The image shows a transmission electron microscope (TEM) image of the interaction between the sea bass antiviral peptide Hepvirusin and eel herpesvirus in Example 5 of this invention. The control group consisted of eel herpesvirus, while the experimental group consisted of eel herpesvirus + 12.5 μM Hepvirusin. Detailed Implementation

[0025] The technical solution of the present invention will be further explained and described below with reference to specific embodiments and accompanying drawings.

[0026] Example 1: Preparation of Hepvirusin, an antiviral peptide for sea bass

[0027] Hepvirusin, an antiviral peptide for sea bass, with a purity of over 95%, can be obtained using existing solid-phase chemical synthesis methods. In this embodiment, the Hepvirusin was obtained by Nanjing Genscript Biotech Co., Ltd. using a solid-phase chemical synthesis method, and they provided information on peptide molecular weight, HPLC, and other detection data.

[0028] The amino acid sequence of the antiviral peptide Hepvirusin is as follows:

[0029] Ile-Lys-Cys-Lys-Phe-Cys-Cys-Gly-Cys-Cys-Thr-Pro-Gly-Val-Cys-Gly-Val-Cys-Cys-Arg-Phe, with the molecular formula C 91 H 148 N 26 O 23 The physicochemical parameters of S8 are shown in Table 1.

[0030] Table 1 Physicochemical parameters of the antibacterial peptide Hepvirusin

[0031]

[0032] As shown in Table 1, Hepvirusin has a small molecular weight and good stability; it is a positively charged cationic polypeptide.

[0033] Example 2: Assay of Hepvirusin, an antiviral peptide for sea bass.

[0034] The cytotoxicity of the sea bass antitoxin Hepvirusin was measured using eel ovarian (EO), eel kidney (EK), and eel spleen (ES) cells, as detailed below:

[0035] (1) Collect eel ovary cells (EO), eel kidney cells (EK), and eel spleen cells (ES) in good growth condition, and adjust the cell concentration to 1×10⁻⁶. 5 Cells were dispersed evenly by blowing them into each well of a 96-well cell culture plate. 100 μL of cell suspension was added to each well of the plate and the plate was incubated at a suitable temperature until more than 80% of the cells adhered to the plate.

[0036] (2) Carefully aspirate the culture medium and add culture medium containing different concentrations (0μM, 0.5μM, 1μM, 6.25μM, 12.5μM, 25μM, 50μM) of Hepvirusin. Incubate in a suitable temperature incubator for 24 hours.

[0037] (3) After adding 20 μL of MTS-PMS solution and incubating in the dark for 2 h, the OD was measured using a microplate reader. 492 The value was used to evaluate the cytotoxicity of Hepvirusin.

[0038] The results are as follows Figure 1 As shown, at a concentration of 50 μM, Hepvirusin showed no toxicity to eel ovarian cells (EO), eel kidney cells (EK), and eel spleen cells (ES).

[0039] Example 3: Determination of the antiviral activity of the sea bass antiviral peptide Hepvirusin against eel herpesvirus

[0040] The virus strain involved in this embodiment is: Fujian strain of eel herpesvirus (AngHV-FJ), which was preserved by the Institute of Biotechnology, Fujian Academy of Agricultural Sciences.

[0041] (1) The Hepvirusin powder synthesized in Example 1 was dissolved in sterile ddH2O and serially diluted to 3.1 μM, 6.3 μM and 12.5 μM, and placed on ice for later use.

[0042] (2) Different concentrations of Hepvirusin were mixed with the virus and added to the cells. A negative control group and a test group were set up, with three replicates in each group.

[0043] (3) After culturing the cells for 24 hours, total DNA was extracted using a blood / cell / tissue genomic DNA extraction kit and used as a template to detect viral load using the established qPCR method.

[0044] The results are as follows Figure 2 As shown, when the concentration reaches 12.5 μM, Hepvirusin can inhibit eel herpesvirus by more than 50%.

[0045] Example 4: One-step growth curve of the antiviral peptide Hepvirusin for sea bass

[0046] The replication of eel herpesvirus was measured to evaluate the inhibitory effect of Hepvirusin on viral replication.

[0047] The specific method is similar to the antiviral activity assay described in Example 3.

[0048] Cells were infected with a mixture of Hepvirusin and virus (final Hepvirusin concentration was 6.3 μM or 12.5 μM). Cells were collected at 3 h, 6 h, 12 h, and 24 h, and total DNA was extracted as a template. Viral load was detected using the established qPCR method. A negative control group and the experimental group were set up, with three replicates for each group.

[0049] The results are as follows Figure 3 As shown, Hepvirusin can inhibit the replication of eel herpesvirus by more than 50% at 24 hours.

[0050] Example 5: Transmission electron microscopy observation of the interaction between the sea bass antiviral peptide Hepvirusin and eel herpesvirus.

[0051] Preparation of eel herpesvirus: The preparation of transmission electron microscopy samples is carried out according to the following steps:

[0052] (1) Dissolve the synthesized peptide Hepvirusin in sterile ddH2O and adjust the peptide concentration to 12.5 μM. Place on ice for later use.

[0053] (2) After mixing the virus solution and Hepvirusin, incubate at 27°C for 60 min.

[0054] (3) The sample was sent to the electron microscopy room for fixation and staining. After drying, it was observed and photographed using a transmission electron microscope.

[0055] The results are as follows Figure 4As shown, the virus in the control group had a normal morphology and intact structure; the virus treated with the antiviral peptide Hepvirusin underwent significant morphological changes, and the envelope ruptured, resulting in leakage of its contents.

[0056] The above description is merely a preferred embodiment of the present invention, and therefore should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made in accordance with the scope of the patent and the contents of the specification should still fall within the scope of the present invention.

Claims

1. The application of the sea bass antiviral peptide Hepvirusin in the preparation of an anti-eel herpesvirus composition, characterized in that: The amino acid sequence of the sea bass antiviral peptide Hepvirusin is shown in SEQ ID NO.1.

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