Application of cortisol in prevention and treatment of aquatic virus infection

Abstract

The invention discloses application of cortisol in prevention and treatment of infection of cyprinid herpesvirus II (CyHV-2), spring viraemia of carp (SVCV) and grass carp reovirus (GCRV), results of a cell line infection model show that the cortisol can play a significant role in protecting cells infected by the CyHV-2, the SVCV and the GCRV, and the cortisol can play a significant role in preventing and treating the infection of the cyprinid herpesvirus II, the SVCV and the GCRV, so that the cortisol can be used for preventing and treating the infection of the cyprinid herpesvirus II, the SVCV and the GCRV, and the application of the cortisol in the prevention and treatment of the infection of the cyprinid herpesvirus II, the SVCV and the GCRV. Furthermore, a molecular biology experiment result shows that the transcription level of the virus related genes treated by cortisol is obviously inhibited. Therefore, cortisol 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 cortisol in the preparation of a drug or additive for preventing or treating aquatic viral infections (especially CyHV-2, SVCV or GCRV). Background Art

[0002] Cortisol is an important glucocorticoid, which has functions such as regulating sugars and proteins, improving blood sugar, and anti-inflammatory effects. The physiological functions of cortisol mainly include: (1) Cortisol can inhibit the aggregation of inflammatory cells such as macrophages and white blood cells at the site of inflammation, thereby reducing the synthesis and release of inflammatory mediators such as prostaglandins and leukotrienes, avoiding the stimulation of the body by inflammatory mediators, and playing an anti-inflammatory role; (2) Cortisol can reduce the stimulatory response of endotoxins in cells, reduce the damage caused by stimulation to cells, and play an antitoxin role; (3) Cortisol can dilate spasmodic and constricted blood vessels, and can also strengthen the contractility of the heart to increase the blood supply and oxygen supply to the body; (4) Cortisol can promote gluconeogenesis, reduce the rate of glucose decomposition, reduce the utilization of glucose by body tissues and increase blood sugar, and can also increase the decomposition and metabolism of proteins, increase the excretion of amino acids and nitrogen, and achieve negative nitrogen balance. At present, the role played by cortisol in the antiviral process is still unclear, and there has been no report on the application of cortisol in the prevention and treatment of viral diseases in aquatic animals.

[0003] Cyprinid Herpesvirus type II (CyHV-2) is the pathogen causing viral gill hemorrhage in cyprinid fish such as goldfish and crucian carp. Once an outbreak occurs, it will cause large-scale deaths of farmed fish. Among them, crucian carp, as an important bulk freshwater economic fish in China, the prevalence of herpesvirus brings huge economic losses every year. Herpesvirus has the characteristics of fast onset and high lethality. At present, the prevention and treatment methods for viral diseases in aquatic animals are relatively limited. Although some studies have been carried out on the pathogen isolation, histopathological observation and transcriptome analysis of fish herpesvirus, no effective prevention and treatment methods have been found. Deeply understanding the pathogen CyHV-2 and proposing effective prevention and treatment measures to control virus outbreaks and reduce economic losses are urgent problems to be solved in the aquaculture industry.

[0004] Spring viraemia of carp virus (SVCV) belongs to the family Rhabdoviridae and the genus Vesiculovirus, and is a single-stranded negative-sense RNA virus. Spring viraemia of carp (SVC) caused by SVCV is a highly contagious and lethal aquatic disease, which can cause huge economic losses. At present, there are relatively rich studies on SVCV, and there are many drug studies, but there are still lack of effective prevention and control measures, and there is no commercial specific drug. In terms of prevention, the vaccine development of SVCV is still in the exploratory stage, and no vaccine that meets the requirements of large-scale production in terms of protection rate, cost, and inoculation method and can be put on the market has been developed. Therefore, under the current research background, finding highly effective and safe drugs is still the first choice for preventing and controlling SVCV.

[0005] Grass carp reovirus (GCRV) belongs to the family Reoviridae and the genus Aquareovirus. Grass carp hemorrhage disease caused by GCRV is the main viral disease that endangers the grass carp aquaculture industry. At present, the prevention and control of GCRV mainly focuses on vaccine research. With the development of the research on genetically engineered vaccines, various grass carp hemorrhage disease vaccines represented by subunit vaccines and nucleic acid vaccines have emerged one after another. However, the large-scale production and application of vaccines are still restricted because the strong specificity of vaccines makes it difficult to apply them to viruses that are prone to mutation. Therefore, finding anti-GCRV drugs with good antiviral effects has obvious advantages compared with using vaccines. Summary of the Invention

[0006] The purpose of the present invention is to provide the application of cortisol in preventing and controlling aquatic virus infections. Cortisol 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 cortisol plays a protective role against CyHV-2, SVCV, and GCRV infections. Cortisol can be used to prepare drugs or additives for preventing and controlling 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 cortisol in preventing and treating aquatic virus infections: In a specific embodiment of the present invention, after treatment with 10 mM cortisol, the cytopathic effect caused by the virus was significantly inhibited and the virus titer was significantly reduced 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 cortisol 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 were significantly decreased, thereby inhibiting virus replication. Animal experiments showed that cortisol could increase the survival rate of zebrafish infected with SVCV.

[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 cells and detecting the transcriptional levels of virus-related genes, it can be known that cortisol is applicable to the prevention or treatment of aquatic viruses (especially CyHV-2, SVCV, or GCRV).

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

[0011] Figure 1 Shows the effects of cortisol on CyHV-2, SVCV, and GCRV-infected cells.

[0012] Figure 2 Shows the effects of cortisol on the transcriptional levels of CyHV-2, SVCV, and GCRV virus genes in cells.

[0013] Figure 3 Shows the effects of cortisol on the lethality rate of zebrafish infected with SVCV. Detailed Embodiments

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

[0015] The antiviral experiment was used to detect the titers of CyHV-2, SVCV and GCRV in cells after cortisol treatment. It was carried out in 24-well cell culture plates and 96-well cell culture plates. GiCB cells were used to detect the virus titer of CyHV-2, EPC cells were used to detect the virus titer of SVCV, and CIK cells were used to detect the virus titer of GCRV. 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, cortisol was added at a final concentration of 10 mM for treatment; after inoculation, the cells were cultured for another 48 h, the cytopathic effect was observed, and the cells were fixed with 4% paraformaldehyde overnight 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 cortisol untreated group and the treated group were calculated according to Reed-Muench. The results are as Figure 1 shown. In the cells infected with CyHV-2, SVCV or GCRV after cortisol treatment, the cytopathic effect was significantly reduced and the virus titer was significantly decreased.

[0016] Example 2: Effects of cortisol on the viral gene transcription of CyHV-2, SVCV and GCRV

[0017] The transcriptional levels of virus-related genes in cells infected with CyHV-2, SVCV or GCRV were detected after treatment with cortisol at final concentrations of 0.4 mM, 1 mM and 0.2 mM respectively. 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 total cell RNA was extracted after lysing the cell pellet with TRIzol (Invitrogen), and cDNA was reverse transcribed by 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, GCRV-related genes s5, s6, s8, s9, and the quantitative primer sequences are shown in Table 1. The results are as Figure 2As shown, after cortisol treatment, 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 cells decreased significantly, thereby inhibiting virus replication.

[0018] Table 1 Quantitative primer sequences

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[0020]

[0021] Example 3: Effect of cortisol on the lethality rate of zebrafish infected with SVCV

[0022] It was carried out in an aquarium of 35 cm × 23.5 cm × 36 cm, and the water was changed daily and the water temperature was maintained at about 22°C. A healthy zebrafish weighing about 1 g was intraperitoneally injected with SVCV virus suspension to establish an artificial infection model. The control group and the experimental group were intraperitoneally injected with 5 μL / fish (5×10 9 TCID 50 / mL) of SVCV virus suspension, and cortisol was diluted in the water of the experimental group to a final concentration of 0.33 μM. The death of zebrafish was recorded daily after infection, and the statistics were carried out until 7 dpi (day post infection). Finally, the Mantel-Cox analysis was used to analyze the survival rate of the fish body ( Figure 3 ). The results showed that the survival rate of the control group after infection with SVCV was only 30%, and the survival rate of the experimental group could be increased to 50% by soaking with cortisol at a final concentration of 0.33 μM. It can be seen that cortisol has an obvious protective effect on zebrafish infected with SVCV.

Claims

1. The use of cortisol 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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