A liquid medicament for the control of viral infections in chickens

Abstract

This invention provides a liquid drug for the prevention and treatment of viral infections in chickens, belonging to the field of veterinary medicine. The liquid drug uses a polypeptide from chicken cartilage as its active ingredient, with the amino acid sequence Gly-Glu-Glu-Gly-Thr-Met-Gly-Leu, and is prepared into solutions with concentrations ranging from 6.25 μg / mL to 100 μg / mL using PBS buffer as a solvent. Experimental results show that this drug can significantly reduce cell death and chicken embryo mortality caused by viral infection by preventing H9N2 virus infection and inhibiting viral replication in host cells, and prolong the survival time of infected individuals. The liquid drug provided by this invention is highly effective, safe, and easy to use, and is suitable for the prevention and control of viral infections in chicken flocks in large-scale poultry farms.

Description

Technical Field

[0001] This invention belongs to the field of animal medicine technology, and in particular relates to a liquid drug for the prevention and treatment of viral infections in chickens. Background Technology

[0002] Avian influenza (AI) is a highly contagious disease caused by type A influenza viruses, widely present in poultry and wild birds, posing a serious threat to the global poultry industry and public health. Among them, the H9N2 subtype of avian influenza virus is one of the most prevalent low-pathogenic avian influenza viruses. This virus can lead to a significant decline in the production performance of chicken flocks, manifested as reduced egg production, stunted growth, and immunosuppression. More importantly, the H9N2 subtype of avian influenza virus has the potential for genetic recombination, enabling it to exchange gene segments with other highly pathogenic avian influenza viruses (such as H5N1 and H7N9), potentially generating new highly pathogenic strains and further exacerbating the risk of disease transmission.

[0003] In terms of treatment, although some antiviral drugs (such as amantadine and oseltamivir) are used to control influenza virus infection, these drugs are mostly designed for human influenza viruses and have limited effectiveness against avian viruses. Furthermore, long-term use can easily lead to drug resistance. Therefore, developing a highly effective, safe, and easy-to-use liquid medication has become a pressing technical challenge in the field of veterinary medicine.

[0004] In recent years, with the deepening of research on natural products, marine organisms, due to their unique ecological environment and rich chemical diversity, have gradually become an important source for the development of new drugs. Squirrel cartilage polypeptide is a bioactive polypeptide extracted from squirrels, with the amino acid sequence Gly-Glu-Glu-Gly-Thr-Met-Gly-Leu. Existing research indicates that this bioactive polypeptide has anti-angiogenic and anti-tumor effects; however, its antiviral effects have not yet been investigated. Summary of the Invention

[0005] The purpose of this invention is to provide a liquid drug for preventing and treating chicken viral infections, thereby providing a new drug for the prevention and treatment of avian influenza virus infection in chickens and expanding the new functions of ray cartilage polypeptide.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] First, the present invention provides a liquid medicine for preventing and treating chicken virus infection, the liquid medicine containing ray cartilage polypeptide as an active ingredient, the amino acid sequence of which is Gly-Glu-Glu-Gly-Thr-Met-Gly-Leu;

[0008] The chicken virus in question is the H9N2 subtype of avian influenza virus.

[0009] Preferably, in the liquid drug, the concentration of the ray cartilage polypeptide is from 6.25 μg / mL to 100 μg / mL.

[0010] Preferably, the liquid drug uses PBS as a solvent.

[0011] Preferably, the ray cartilage polypeptide achieves the effect of preventing and controlling H9N2 subtype avian influenza virus infection by preventing H9N2 subtype avian influenza virus infection and inhibiting H9N2 subtype avian influenza virus infection replication.

[0012] Preferably, the H9N2 subtype avian influenza virus strain is an F98-like strain.

[0013] Secondly, this invention provides the application of ray cartilage polypeptide in the preparation of a liquid drug for preventing infection with chicken H9N2 subtype avian influenza virus, wherein the amino acid sequence of the ray cartilage polypeptide is Gly-Glu-Glu-Gly-Thr-Met-Gly-Leu.

[0014] Preferably, in the liquid drug, the concentration of the ray cartilage polypeptide is from 25 μg / mL to 100 μg / mL.

[0015] Preferably, the H9N2 subtype avian influenza virus strain is an F98-like strain.

[0016] Furthermore, this invention provides the application of ray cartilage polypeptide in the preparation of a liquid drug for inhibiting H9N2 subtype avian influenza virus infection, characterized in that the amino acid sequence of the ray cartilage polypeptide is Gly-Glu-Glu-Gly-Thr-Met-Gly-Leu.

[0017] Preferably, in the liquid drug, the concentration of the ray cartilage polypeptide is from 6.25 μg / mL to 100 μg / mL.

[0018] Preferably, the H9N2 subtype avian influenza virus strain is an F98-like strain.

[0019] Finally, this invention provides the application of the ray cartilage polypeptide in the preparation of a drug for treating chicken embryo death caused by H9N2 subtype avian influenza virus infection, wherein the amino acid sequence of the ray cartilage polypeptide is Gly-Glu-Glu-Gly-Thr-Met-Gly-Leu;

[0020] The concentration of the ray cartilage polypeptide was 100 μg / mL.

[0021] Preferably, the H9N2 subtype avian influenza virus strain is an F98-like strain.

[0022] The beneficial effects of this invention are as follows:

[0023] Previous research on ray cartilage peptides has mainly focused on anti-angiogenesis and anti-tumor applications, but this invention reveals a novel function in the antiviral field for the first time. Through in vitro and in vivo experiments, ray cartilage peptides have been shown to significantly inhibit the infection and replication process of H9N2 subtype avian influenza virus, a discovery that opens up new avenues for its application in veterinary medicine.

[0024] The experimental results of this invention show that the keratinocyte chondrocyte polypeptide can not only prevent H9N2 virus infection, but also effectively inhibit the replication stage after viral infection. This multi-mechanism synergistic effect makes the keratinocyte chondrocyte polypeptide show unique advantages in the field of antiviral treatment, thereby supplementing existing vaccine strategies.

[0025] Meanwhile, the results of this invention show that the cartilage polypeptide of the ray has a certain specificity for H9N2 virus infection, thereby enabling precise prevention and control of H9N2 virus. Attached Figure Description

[0026] Figure 1 The results of cytotoxicity assay of keratinocyte chondrocyte polypeptide against DF-1 cells;

[0027] Figure 2 The inhibitory effect of ray cartilage peptides on the replication of H9N2 subtype avian influenza virus infection;

[0028] Figure 3 The inhibitory effect of ray cartilage peptides on Newcastle disease virus replication;

[0029] Figure 4 The results of the detection of the direct killing effect of ray cartilage polypeptide on H9N2 subtype avian influenza virus;

[0030] Figure 5 The results show the preventive effect of ray cartilage peptides on H9N2 subtype avian influenza virus infection. Detailed Implementation

[0031] Example 1

[0032] To evaluate the cytotoxicity of the keratinocyte chondrocyte polypeptide (synthesized by Shanghai Newp Biotechnology, with a purity of 97.6%) on DF-1 cells (chicken fibroblasts), this experiment was conducted according to the following steps to determine the safe concentration range of the polypeptide and provide basic data for subsequent antiviral activity studies.

[0033] 1. Seed DF-1 cells into 96-well plates at a density of approximately 1×10^4 cells / well. Incubate the seeded 96-well plates in a constant temperature incubator at 37°C and 5% CO2 until the cells adhere and reach 80%-90% confluence.

[0034] 2. A series of concentration gradients of ray cartilage polypeptide solutions were prepared using DMEM-F12 medium (containing 10% fetal bovine serum) at concentrations of 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL and 6.25 μg / mL.

[0035] 3. Add 100 μL of peptide solution of different concentrations to each well, and set 5 replicates for each concentration to improve the reliability of the experimental results. The control group is only added with an equal amount of DMEM-F12 medium, without adding peptides.

[0036] 4. Place the 96-well plate back into the incubator at 37°C and 5% CO2 and continue culturing for 48 hours.

[0037] 5. After the culture is completed, add 10 μL of CCK-8 solution to each well, mix gently, and continue incubation for 3 hours. Then, use a microplate reader to measure the absorbance (OD value) of each well at a wavelength of 450 nm and calculate the cell viability (%).

[0038] Table 1. Cell viability test results

[0039]

[0040] From Table 1 and Figure 1 The experimental results showed that there was no significant difference in cell survival rate between the experimental groups and the control group, indicating that the polypeptide had no obvious toxic effect on cells within the tested concentration range.

[0041] Example 2

[0042] Detecting the inhibitory effect of ray cartilage peptides on the replication of H9N2 subtype avian influenza virus infection.

[0043] 1. DF-1 cells were seeded into 96-well plates using DMEM-F12 medium (containing 10% fetal bovine serum) at a density of approximately 1×10^4 cells / well. The seeded 96-well plates were then placed in a constant temperature incubator at 37°C and 5% CO2 until the cells adhered and reached 95% confluence.

[0044] 2. Remove the culture medium, add 100 TCID50 / 0.1 mL of H9N2 subtype avian influenza virus solution (F98-like strain) to each well, and incubate for 2 hours.

[0045] 3. After incubation, discard the virus solution and wash three times with PBS buffer to remove unadsorbed virus particles. Then, add 100 μL of different concentrations of ray cartilage peptide solution (6.25 μg / mL, 25 μg / mL, 100 μg / mL) to each well. A control group was also set up as follows:

[0046] Normal control group: Contains only cells and culture medium, used to assess the growth status of cells under normal culture conditions;

[0047] Virus control group: containing only cells and virus fluid, without added peptides, used to determine the effect of virus acting alone on cells;

[0048] Blank control group: containing only culture medium, without cells and viruses, serving as a reference for the experimental background and excluding interference factors that may be introduced during the experimental procedure;

[0049] The 96-well plate was returned to an incubator at 37°C and 5% CO2 and incubated for another 48 hours.

[0050] After the culture was completed, 10 μL of CCK-8 solution was added to each well, gently mixed, and incubated for another 3 hours. Then, the absorbance (OD value) of each well was measured at a wavelength of 450 nm using a microplate reader, and the cell viability (%) was calculated.

[0051] Table 2. Inhibitory effect of ray cartilage peptides on replication of H9N2 subtype avian influenza virus infection.

[0052]

[0053] From Table 2 and Figure 2 The experimental results showed that, compared with the normal control group (cell survival rate of 100%), the cell survival rate of the virus control group was significantly reduced, indicating that the H9N2 subtype avian influenza virus successfully infected DF-1 cells and caused significant cell damage. This result verifies the successful construction of the virus infection model in this study and provides a reliable experimental basis for subsequent evaluation of the antiviral activity of the ray cartilage peptide.

[0054] Further analysis revealed that, compared to the virus control group, treatment with three different concentrations (6.25 μg / mL, 25 μg / mL, and 100 μg / mL) of *Pterygium argentide* cartilage peptide significantly improved cell viability in a concentration-dependent manner. These results indicate that *Pterygium argentide* cartilage peptide significantly inhibits the replication of H9N2 subtype avian influenza virus, effectively mitigating viral damage to cells and protecting them from virus-induced cell death.

[0055] Example 3

[0056] Detecting the inhibitory effect of ray cartilage peptides on Newcastle disease virus replication.

[0057] 1. Seed DF-1 cells into 96-well plates at a density of approximately 1×10^4 cells / well. Incubate the seeded 96-well plates in a constant temperature incubator at 37°C and 5% CO2 until the cells adhere and reach 95% confluence.

[0058] 2. Remove the culture medium and add 100 TCID50 / 0.1 mL of Newcastle disease virus NA-1 strain solution (strain number: CGMCC9992) to each well, and incubate for 2 hours.

[0059] 3. After incubation, discard the virus solution and wash three times with PBS buffer to remove unadsorbed virus particles. Then, add 100 μL of different concentrations of ray cartilage peptide solution (6.25 μg / mL, 25 μg / mL, 100 μg / mL) to each well.

[0060] The following control group was also set up:

[0061] Normal control group: No virus treatment was performed, but the medium was replaced with 5% FBS DMEM.

[0062] Virus control group: only virus solution was treated and the medium was replaced with 5% FBS DMEM;

[0063] Blank control group: containing only 5% FBS DMEM medium, without cells and viruses;

[0064] The 96-well plate was returned to an incubator at 37°C and 5% CO2 and incubated for another 48 hours.

[0065] 4. After the culture is completed, add 10 μL of CCK-8 solution to each well, mix gently, and continue incubation for 3 hours. Then, use a microplate reader to measure the absorbance (OD value) of each well at a wavelength of 450 nm and calculate the cell viability (%).

[0066] Table 3. Inhibitory effect of ray cartilage peptides on Newcastle disease virus infection.

[0067]

[0068] From Table 3 and Figure 3The experimental results showed that, compared with the normal control group, the cell survival rate of the virus control group was significantly reduced, indicating that Newcastle disease virus successfully infected DF-1 cells and caused significant damage to the cells. However, compared with the virus control group, the cell survival rate did not increase significantly after treatment with keratinocyte chondrocyte peptides, indicating that the virus inhibitory effect of keratinocyte chondrocyte peptides is specific and cannot inhibit infection caused by Newcastle disease virus. This may be related to the different infection and replication modes of Newcastle disease virus and H9N2 subtype avian influenza virus.

[0069] Example 4

[0070] Detection of the direct killing effect of ray cartilage peptides on H9N2 subtype avian influenza virus.

[0071] 1. Seed DF-1 cells into 96-well plates at a density of approximately 1×10^4 cells / well. Incubate the seeded 96-well plates in a constant temperature incubator at 37°C and 5% CO2 until the cells adhere and reach 95% confluence.

[0072] 2. H9N2 virus solution was mixed with different concentrations of ray cartilage polypeptide solutions to achieve polypeptide concentrations of 6.25 μg / mL, 25 μg / mL, and 100 μg / mL, while maintaining a viral load of 100 TCID. 50 / 0.1mL, blended at 37℃ for 4h.

[0073] 3. Add 100 μL of the blended solution and 100 μL of the untreated virus solution to 96-well plates and incubate for 2 h.

[0074] 4. After incubation, discard the virus solution and wash three times with PBS buffer to remove unadsorbed virus particles, then replace with 5% FBS DMEM medium;

[0075] The following control group was also set up:

[0076] Normal control group: No virus treatment was performed, but the medium was replaced with 5% FBS DMEM.

[0077] Virus control group: only virus solution was treated and the medium was replaced with 5% FBS DMEM;

[0078] Blank control group: containing only 5% FBS DMEM medium, without cells and viruses;

[0079] The 96-well plate was returned to an incubator at 37°C and 5% CO2 and incubated for another 48 hours.

[0080] 5. After the culture is completed, add 10 μL of CCK-8 solution to each well, mix gently, and continue incubation for 3 hours. Then, use a microplate reader to measure the absorbance (OD value) of each well at a wavelength of 450 nm and calculate the cell viability (%).

[0081] Table 4. Direct killing effect of ray cartilage peptides on H9N2 subtype avian influenza virus.

[0082]

[0083] From Table 4 and Figure 4 The experimental results show that, compared with the virus control group, directly mixing different concentrations of ray cartilage polypeptide with H9N2 subtype avian influenza virus does not directly kill H9N2 subtype avian influenza virus.

[0084] Example 5

[0085] Detection of the preventive effect of ray cartilage peptides on H9N2 subtype avian influenza virus infection.

[0086] 1. Seed DF-1 cells into 96-well plates at a density of approximately 1×10^4 cells / well. Incubate the seeded 96-well plates in a constant temperature incubator at 37°C and 5% CO2 until the cells adhere and reach 95% confluence.

[0087] 2. Remove the culture medium and add 100 μL of different concentrations of ray cartilage polypeptide solution (6.25 μg / mL, 25 μg / mL, 100 μg / mL) to each well for 12 h of pretreatment culture;

[0088] 3. Remove the culture medium, add 100 TCID50 / 0.1 mL of H9N2 subtype avian influenza virus to each well, and incubate for 2 hours;

[0089] 4. After incubation, discard the virus solution, wash three times with PBS buffer, and replace with 5% FBS DMEM medium;

[0090] The following control group was also set up:

[0091] Normal control group: No virus treatment was performed, but the medium was replaced with 5% FBS DMEM.

[0092] Virus control group: only virus solution was treated and the medium was replaced with 5% FBS DMEM;

[0093] Blank control group: containing only 5% FBS DMEM medium, without cells and viruses.

[0094] 5. After the culture is completed, add 10 μL of CCK-8 solution to each well, mix gently, and continue incubation for 3 hours. Then, use a microplate reader to measure the absorbance (OD value) of each well at a wavelength of 450 nm and calculate the cell viability (%).

[0095] Table 5. Preventive effect of ray cartilage peptides on H9N2 subtype avian influenza virus.

[0096]

[0097] From Table 5 and Figure 5 The experimental results showed that although cell viability was slightly improved after pretreatment with 6.25 μg / mL of keratinocyte chondrocyte polypeptide (compared to the virus control group), the difference was not statistically significant (P>0.05). This indicates that at this concentration, the preventive effect of keratinocyte chondrocyte polypeptide against H9N2 virus infection is limited and may not be sufficient to achieve the desired preventive effect.

[0098] Compared with the virus control group, the cell survival rate was significantly increased after treatment with 25 μg / mL and 100 μg / mL of ray cartilage peptide, indicating that pretreatment of cells with the above concentrations of ray cartilage peptide can effectively prevent infection with H9N2 subtype avian influenza virus.

[0099] Example 6

[0100] Detection of the therapeutic effect of ray cartilage peptides on chicken embryos infected with H9N2 subtype avian influenza virus.

[0101] 1. Select 9-day-old SPF chicken embryos (healthy embryos without other infections), and group and label the embryos according to the following grouping method:

[0102] First, number each chicken embryo above its air cell with a marker, then randomly divide the 45 chicken embryos into 3 groups:

[0103] Normal control group: Inoculated with 100 μl PBS, followed by injection of 100 μl PBS;

[0104] Virus control group: Inoculated with 100 μl of 100 TCID50 virus solution, followed by injection of 100 μl of PBS;

[0105] Peptide treatment group: 100 μl of 100 TCID50 virus solution was inoculated, followed by injection of 100 μl of ray cartilage peptide solution (prepared in PBS, concentration 100 μg / mL).

[0106] 2. Inject the H9N2 virus solution into the allantoic cavity of the chicken embryo using a sterile syringe, seal it with paraffin, and place it in an incubator for further cultivation;

[0107] 3. Two hours after inoculation, according to the experimental group, 100 μl of ray cartilage polypeptide solution was injected into the chicken embryos of the polypeptide treatment group, the embryos were sealed with paraffin, and then placed back into the incubator for continued culture.

[0108] 4. Monitor the survival status of chicken embryos regularly every day. The mortality criteria are as follows:

[0109] Chicken embryos stop developing or show obvious lesions (such as hemorrhage, edema, etc.);

[0110] Shine a flashlight on the chicken embryo; if there is no blood vessel pulsation or embryonic activity;

[0111] Observe continuously for seven days and calculate the chicken embryo mortality rate and average time to death.

[0112] Table 6. Therapeutic effect of ray cartilage peptides on chicken embryos infected with H9N2 subtype avian influenza virus.

[0113]

[0114] As can be seen from the results in Table 6, compared with the virus control group, the keratin peptide used in the peptide treatment group can effectively reduce chicken embryo mortality caused by H9N2 subtype avian influenza virus, and can also effectively delay the average mortality time, fully demonstrating its potential in the treatment and prevention of H9N2 subtype avian influenza virus infection.

Claims

1. The application of ray cartilage polypeptide in the preparation of a liquid drug for preventing infection with chicken H9N2 subtype avian influenza virus, characterized in that, The amino acid sequence of the ray cartilage polypeptide is Gly-Glu-Glu-Gly-Thr-Met-Gly-Leu; In the liquid drug, the concentration of the ray cartilage polypeptide is from 25 μg / mL to 100 μg / mL.

2. The application of ray cartilage polypeptide in the preparation of a liquid drug for inhibiting H9N2 subtype avian influenza virus infection, characterized in that, The amino acid sequence of the ray cartilage polypeptide is Gly-Glu-Glu-Gly-Thr-Met-Gly-Leu; In the liquid drug, the concentration of the ray cartilage polypeptide is from 6.25 μg / mL to 100 μg / mL.

3. The application of ray cartilage polypeptide in the preparation of a drug for treating chicken embryo mortality caused by H9N2 subtype avian influenza virus infection, characterized in that... The amino acid sequence of the ray cartilage polypeptide is Gly-Glu-Glu-Gly-Thr-Met-Gly-Leu; The concentration of the ray cartilage polypeptide was 100 μg / mL.

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