Application of lactic acid in prevention and treatment of aquatic virus infection

Abstract

The invention discloses an application of lactic acid (Lactate) in prevention and treatment of infection of CyHV-2 (Cyprinid Herpesvirus, CyHV-2), SVCV (Spring Viraemia of Carp Virus) and GCRV (Grass Carp Reovirus), a result of a cell line infection model shows that the lactic acid has a significant protection effect on cells infected by CyHV-2, SVCV and GCRV, and the lactic acid has a significant protection effect on cells infected by CyHV-2, SVCV and GCRV. Furthermore, a molecular biology experiment result shows that the transcription level of the virus related genes treated by lactic acid is obviously inhibited. Therefore, the lactic acid can be used as a medicine or an additive for preventing and treating CyHV-2, SVCV and GCRV infection, and has the advantages of low dosage and good protection effect.

Description

Technical Field

[0001] The present invention belongs to the technical field of aquaculture, and particularly relates to the use of lactic acid in preparing a drug or additive for preventing or treating aquatic virus (especially CyHV-2, SVCV or GCRV) infections. Background Art

[0002] Lactate is an important organic acid and the main product of anaerobic or aerobic glycolysis. It participates in a variety of physiological processes and metabolic pathways and has broad biocompatibility. The physiological functions of lactate mainly include: (i) an intermediate in energy metabolism, lactate plays an important role in cell metabolism, especially under hypoxic conditions, serving as an energy substrate to support the energy requirements of cells; (ii) promoting muscle growth and repair, lactate can stimulate the proliferation and differentiation of myoblasts, promote muscle regeneration, and enhance the muscle repair ability; (iii) regulating immune function, lactate, as an immune system regulator, affects the functions of immune cells in inflammatory diseases and participates in the regulation of immune responses by influencing the activity and functions of immune cells; (iv) maintaining intracellular signal transduction, lactate participates in the post-translational modification of proteins through lactylation reactions, affects cell signal transduction and gene expression, and thus plays a role in the occurrence and development of diseases. Although the application of lactate in the prevention and treatment of viral diseases in aquatic animals is still in the research stage, its role in regulating immune responses and cell signal transduction provides potential application prospects for the prevention and treatment of viral diseases in aquatic animals.

[0003] Cyprinid Herpesvirus type II (CyHV-2) is an important pathogen in the aquaculture industry and poses a serious threat to cyprinid fish, especially goldfish and crucian carp. The diseases caused by CyHV-2 are highly contagious and have a high lethality rate, which has a great impact on the fish farming industry. Due to the lack of effective treatment methods and commercial vaccines at present, the prevention and control of CyHV-2 have become an important challenge faced by the aquaculture industry. Therefore, strengthening the monitoring and research of CyHV-2 and developing effective preventive measures are crucial for ensuring the sustainable development of the aquaculture industry.

[0004] Spring viraemia of carp virus (SVCV) is a pathogen that has a significant impact on the aquaculture industry. It belongs to the family Rhabdoviridae and the genus Vesiculovirus. The spring viraemia of carp (SVC) caused by it is an acute, hemorrhagic, highly contagious and highly lethal disease. SVCV mainly affects cyprinid fish, especially carp and goldfish with high economic value, and can cause a mortality rate of over 90%. The virus breaks out when the water temperature is relatively low in spring, bringing huge economic losses to the global aquaculture industry. Due to the lack of effective treatment methods and commercial vaccines, the prevention and control of SVCV have become an important challenge for the aquaculture industry. Therefore, strengthening the monitoring and research of SVCV and developing effective preventive measures are crucial for ensuring the sustainable development of the aquaculture industry.

[0005] Grass carp reovirus (GCRV) is an important pathogen in the aquaculture industry, mainly affecting grass carp (Ctenopharyngodon idella). GCRV belongs to the family Reoviridae and the genus Aquareovirus, and is a double-stranded RNA virus with strong pathogenicity. The hemorrhagic disease caused by this virus has brought serious economic losses to the grass carp aquaculture industry, especially in Asian countries such as China, North Korea and Vietnam. Grass carp infected with GCRV will show symptoms such as congestion and bleeding in muscles, intestines, fins and gills, leading to large-scale death. Due to the lack of effective treatment methods, preventive measures such as vaccination and the use of immunopotentiators have become the main methods to control the spread of GCRV. However, the large-scale production and application of vaccines are still restricted, mainly because the strong specificity of vaccines makes them unable to be applied to easily mutated viruses. Therefore, developing broad-spectrum, highly efficient and environmentally friendly anti-GCRV drugs is an urgent problem to be solved in the aquaculture industry. Summary of the Invention

[0006] The purpose of the present invention is to provide the application of lactic acid in preventing and treating aquatic virus infections. Lactic acid can significantly inhibit the transcription of virus-related genes (CyHV-2-related genes orf60, orf72, orf80, orf92, SVCV-related genes n, p, l, m and GCRV-related genes s5, s6, s8, s9) in fish in vitro cultured cells. Antiviral experiments have confirmed that lactic acid plays a protective role against CyHV-2, SVCV and GCRV infections. Lactic acid can be used to prepare drugs or additives for preventing and treating aquatic virus (especially CyHV-2, SVCV and GCRV) infections.

[0007] In order to achieve the above purpose, the present invention adopts the following technical solutions:

[0008] Application of lactic acid in preventing and treating aquatic virus infections: In specific embodiments of the present invention, after treatment with lactic acid at a final concentration of 10 mM, the cytopathic effects caused by the virus were significantly inhibited in the GiCB cell line (Gibel carp brain cells) infected with CyHV-2, the EPC cell line (epithelioma papulosum cyprinid cells) infected with SVCV, or the CIK cell line (Ctenopharyngodon idellus kidney cells) infected with GCRV. Further detection of the transcriptional levels of virus-related genes in the cells showed that after lactic acid treatment, the transcriptional levels of virus-related genes (CyHV-2-related genes orf60, orf72, orf80, orf92, SVCV-related genes n, p, l, m, and GCRV-related genes s5, s6, s8, s9) in GICB, EPC, and CIK cells decreased significantly, thus inhibiting virus replication. Animal experiments showed that lactic acid could significantly improve the survival rates of zebrafish infected with SVCV and crucian carp infected with CyHV-2.

[0009] In the embodiments of the present invention, the three fish cell lines selected are cell lines derived from the main hosts of CyHV-2, SVCV, and GCRV. Therefore, by establishing disease models of virus infection in three cell lines and detecting the expression of virus-related genes, it can be known that lactic acid is applicable to the prevention and treatment of aquatic viruses (especially CyHV-2, SVCV, or GCRV).

[0010] Lactic acid can be used to prepare drugs or additives for preventing or treating infections with CyHV-2, SVCV, or GCRV. The drugs can be used in the form of injections or oral preparations, and have the advantages of low dosing, single component, and good prevention and treatment effects. Brief Description of the Drawings

[0011] Figure 1 Effects of lactic acid on CyHV-2, SVCV, and GCRV-infected cells.

[0012] Figure 2 Effects of lactic acid on the transcriptional levels of CyHV-2, SVCV, and GCRV virus genes in cells.

[0013] Figure 3 Effects of lactic acid on the lethality rates of zebrafish infected with SVCV and crucian carp infected with CyHV-2. Detailed Description of the Embodiments

[0014] Example 1: Effects of lactic acid on CyHV-2, SVCV, and GCRV-infected cells

[0015] The antiviral experiment was used to detect the titers of CyHV-2, SVCV and GCRV viruses in cells after lactic acid treatment. It was carried out in 24-well cell culture plates and 96-well cell culture plates. GiCB cells were used to detect the titer of CyHV-2 virus, EPC cells were used to detect the titer of SVCV virus, and CIK cells were used to detect the titer of GCRV virus. Cells in good growth state were passaged and seeded into 24-well plates and cultured overnight at 28 °C; CyHV-2 with an MOI of 0.0001 was inoculated into GiCB cells, SVCV with an MOI of 1 was inoculated into EPC cells, and GCRV with an MOI of 0.001 was inoculated into CIK cells, and at the same time lactic acid with a final concentration of 10 mM was added for treatment; after inoculation, the cells were continuously cultured for 48 h, the cytopathic effect was observed, fixed overnight with 4% paraformaldehyde, and stained with 1% crystal violet. The supernatant before cell fixation was collected and stored at 4 °C for detecting the virus titer. The TCID 50 method was used to detect the virus titer: Cells in good growth state were inoculated into 96-well plates and cultured overnight at 28 °C, and cell supernatants with different dilution multiples were inoculated. After 48 h, the formation of plaques in the cells was observed. The virus titers of the lactic acid untreated group and the treated group were calculated according to Reed-Muench. The results are as Figure 1 shown. In virus-infected cells treated with 10 mM lactic acid, the cytopathic effect was significantly reduced and the virus titer was significantly decreased.

[0016] Example 2: Effects of lactic acid on the gene transcription of CyHV-2, SVCV and GCRV viruses

[0017] To detect the transcriptional levels of virus-related genes in cells after cells infected with CyHV-2, SVCV or GCRV were treated with lactic acid at a final concentration of 10 mM. Cells in good growth state were passaged and seeded into 6-well plates and cultured overnight at 28 °C; the virus was inoculated and the drug treatment was carried out according to the method and virus titer described in Example 1; after continuous culture for 48 h, the cell culture medium was aspirated, and the cell precipitate was lysed with TRIzol (Invitrogen) and then the total cellular RNA was extracted. cDNA was reverse transcribed by the GoScript reverse transcription kit (Promega). The transcriptional levels of virus-related genes were detected by qPCR, including CyHV-2-related genes orf60, orf72, orf80, orf92, SVCV-related genes n, p, l, m, and GCRV-related genes s5, s6, s8, s9. The quantitative primer sequences are shown in Table 1. The results are as Figure 2 shown. After treatment with 10 mM lactic acid, the transcriptional levels of CyHV-2-related genes (including orf60, orf72, orf80, orf92), SVCV-related genes (including n, p, l, m) and GCRV-related genes (including s5, s6, s8, s9) in the cells were significantly decreased, thus inhibiting virus replication.

[0018] Table 1 Quantitative primer sequences

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[0021] Example 3: Effects of lactic acid on the lethality of zebrafish infected with SVCV and crucian carp infected with CyHV-2

[0022] It was carried out in an aquarium of 35 cm × 23.5 cm × 36 cm, and the water was changed every day and the water temperature was maintained at about 22 °C. Healthy zebrafish of about 1 g and healthy crucian carp of about 5 g were respectively established as artificial infection models by intraperitoneal injection of SVCV or CyHV-2 virus suspension and the mixed suspension of SVCV or CyHV-2 virus and lactic acid. The control group of zebrafish was intraperitoneally injected with 5 μL / tail of SVCV virus suspension (5×10 9 TCID 50 / mL), and in the experimental group, lactic acid was diluted with SVCV virus suspension to a final concentration of 0.05 mol / L, and at the same time, 5 μL / tail was injected for infection. The control group of crucian carp was intraperitoneally injected with 10 μL / tail of CyHV-2 virus suspension (5×10 9 TCID 50 / mL), and in the experimental group, lactic acid was diluted with CyHV-2 virus suspension to a final concentration of 0.05 mol / L, and at the same time, 10 μL / tail was injected for infection. The death conditions of zebrafish and crucian carp were recorded every day after infection, and were respectively counted until 7 dpi (day post infection) and 14 dpi, and finally the survival rate of fish bodies was analyzed by Mantel-Cox ( Figure 3 ). The results showed that the survival rate of the control group of zebrafish infected with SVCV was only 50%, and the survival rate could be increased to 75% after injecting 0.03 mg / g of lactic acid in total. The survival rate of the control group of crucian carp infected with CyHV-2 was 6.25%, and the survival rate could be increased to 87.5% after injecting 0.009 mg / g of lactic acid in total. It can be seen that lactic acid has an obvious protective effect on zebrafish and crucian carp infected with SVCV and CyHV-2.

Claims

1. Application of lactic acid in the preparation of drugs or additives for preventing and treating aquatic viral infections.

2. The use according to claim 1, characterized in that: The aquatic virus is type II carp herpes virus, carp spring viremia virus or grass carp reovirus.

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