Application of lactate ion in antiviral treatment of fish

Abstract

The present invention belongs to the technical field of aquaculture and aquatic virus prevention and control, and specifically relates to the application of lactate ions in the preparation of products for preventing and treating carp spring viremia. The present invention finds that the lack of lactate dehydrogenase (LDHA) will lead to a decrease in the resistance of zebrafish to SVCV infection, and then concludes that the supplementation of lactate ions can achieve the effect of preventing and treating carp spring viremia virus (SVCV) infection. Specific lactate ions can effectively enhance the fish body's resistance to SVCV infection, inhibit SVCV replication and proliferation, and increase the survival rate of infected fish bodies, and can be effectively applied to SVCV prevention and treatment in carp fish farming. And the sodium lactate that provides lactate ions is low in price and highly economical.

Description

Technical Field

[0001] The invention belongs to the technical field of aquaculture and aquatic virus prevention and control, and particularly relates to application of lactate ions in preparing products for preventing and treating carp spring viremia. Background Art

[0002] Fish pathogenic diseases include bacterial, fungal, viral and parasitic diseases. Viral diseases have a long incubation period and are difficult to detect. Once they occur, they spread quickly and are difficult to control. Spring carp viremia virus (SVCV) is the pathogen of spring carp viremia (SVC) and belongs to the genus Vesiculovirus of the family Rhabdoviridae. Spring carp viremia virus is widely prevalent, causing high morbidity and mortality in aquaculture fish in Europe, America and Asia. Spring carp viremia virus has a wide range of hosts. The main economic fish species in my country, such as black carp, grass carp, silver carp, bighead carp and carp, model organism zebrafish, and ornamental fish koi can all be infected with SVCV. SVCV is usually prevalent in spring, and the water temperature is between 10℃ and 17℃, which is most conducive to the proliferation and spread of SVCV. Fish infected with SVCV show complex symptoms, such as liver and vascular necrosis, inflammatory response, spleen congestion, gill and skin bleeding, and gill plate degeneration.

[0003] Studies have found that SVCV-infected cells can inhibit the ubiquitination modification of p53. Therefore, the existing technology mostly increases the ubiquitination modification of p53 K63 to achieve the effect of inhibiting SVCV, such as through Ca 2+ To regulate p53 to inhibit SVCV and thus prevent carp spring viremia.

[0004] In the metabolic process of animals, under aerobic conditions, hexose in the cytoplasm is broken down into pyruvate, and pyruvate enters the mitochondria to undergo the tricarboxylic acid cycle. Under anaerobic conditions, pyruvate is converted into lactic acid through lactic acid fermentation under the action of lactate dehydrogenase LDHA. Pathogen infection can cause local tissue or cell hypoxia to enhance the production of lactic acid. In the past, lactic acid has been considered a metabolic waste of glucose. However, studies in recent years have found that lactic acid plays a key role in regulating a variety of biological processes, such as macrophage polarization, helper T cell differentiation, and tumor immune monitoring, but the role of lactic acid or lactate in fish antiviral is still unclear. Summary of the invention

[0005] The purpose of the present invention is to provide an application of lactate ions in the preparation of products for preventing and treating carp spring viremia. Lactate ions can inhibit the replication and proliferation of carp spring viremia viruses, improve the survival rate of infected fish, and effectively prevent and treat carp spring viremia in the breeding process of carp fish.

[0006] Application of lactate ion in the preparation of products for preventing and treating carp spring viremia.

[0007] Preferably, the product for preventing and treating carp spring viremia includes one or more products of enhancing the fish body's resistance to carp spring viremia virus infection, inhibiting the replication of carp spring viremia virus infection and inhibiting the proliferation of carp spring viremia virus infection.

[0008] Preferably, the lactate ion is used at a concentration of 1 to 15 mM.

[0009] Preferably, the substance providing the lactate ions includes sodium lactate.

[0010] Preferably, the product comprises medicine or feed.

[0011] Preferably, the dosage form of the drug includes a liquid or a solid dosage form.

[0012] Preferably, the liquid agent includes an injection or an immersion agent.

[0013] Preferably, the feed comprises aquatic feed and a lactate ion component.

[0014] The present invention also provides a product for preventing and treating carp spring viremia, the product comprising a lactate ion component and an auxiliary material; when the product is a medicine, the auxiliary material is a pharmaceutically acceptable auxiliary material; when the product is a feed, the auxiliary material is aquatic feed.

[0015] Beneficial effects:

[0016] The present invention provides the use of lactate ions in the preparation of products for preventing and treating carp spring viremia. The present invention finds that the lack of lactate dehydrogenase (LDHA) will lead to a decrease in the resistance of zebrafish to SVCV infection, and further concludes that the supplementation of lactate ions can achieve the effect of preventing and treating SVCV infection. Specifically, lactate ions can effectively enhance the fish body's resistance to carp spring viremia virus (SVCV) infection, inhibit SVCV replication and proliferation, and increase the survival rate of infected fish bodies, and can be effectively applied to the prevention and treatment of SVCV in carp fish farming. And sodium lactate that provides lactate ions is low in price and highly economical. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0018] Figure 1 This is a graph showing the effects of different concentrations of sodium lactate on the growth and development of zebrafish fry in Example 1;

[0019] Figure 2 The growth and development of zebrafish fry in different test groups in Example 2;

[0020] Figure 3 The fluorescence quantitative expression results of the carp spring viremia virus-related genes in Example 3;

[0021] Figure 4 The growth and development and related gene expression of the lactate dehydrogenase-deficient zebrafish infected with carp spring viremia virus in Example 4. DETAILED DESCRIPTION

[0022] The invention provides application of lactate ions in preparing products for preventing and treating carp spring viremia.

[0023] The product of the present invention preferably includes one or more products of enhancing the fish body's resistance to carp spring viremia virus infection, inhibiting the replication of carp spring viremia virus infection and inhibiting the proliferation of carp spring viremia virus infection, and more preferably includes a product of enhancing the fish body's resistance to carp spring viremia virus infection, inhibiting the replication of carp spring viremia virus infection and inhibiting the proliferation of carp spring viremia virus infection.

[0024] The concentration of lactate ion used in the present invention is preferably 1-15 mM, more preferably 5-10 mM, and more preferably 5 mM. Lactate ion within the concentration range used in the present invention has the effect of preventing and treating carp spring viremia, but the concentration of 5 mM can ensure the effect of preventing and treating carp spring viremia without other serious damage to the fish body.

[0025] The medicine of the present invention preferably includes medicine or feed; the dosage form of the medicine preferably includes liquid or solid; the liquid preferably includes injection or soaking; the feed preferably includes aquatic feed and lactate ion components.

[0026] Specifically, the present invention preferably adopts an immersion method to immerse SVCV infected young fish and / or fry in a solution containing lactate ions for treating infected larvae. The immersion time described in the present invention is preferably 12 to 24 hours, more preferably 12 hours. Alternatively, the present invention preferably adopts an intraperitoneal injection method to inject a solution containing lactate ions into the abdominal cavity of adult fish for treating infected adult fish. Alternatively, the present invention preferably prepares a mixed feed containing lactate ions into fish aquatic feed, and feeds the fish to prevent and / or treat SVCV infection. The amount of lactate ions in the mixed feed described in the present invention is preferably 2.4%. In the present invention, the application objects of the lactate ions preferably include carp.

[0027] The present invention also provides a product for preventing and treating spring viremia in carp, the product comprising a lactate ion component and an auxiliary material; when the product is a drug, the auxiliary material is a pharmaceutically acceptable auxiliary material; when the product is a feed, the auxiliary material is an aquatic feed. The present invention has no special restrictions on the type and amount of the auxiliary material, and it can be conventionally set according to needs.

[0028] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0029] Example 1

[0030] Treatment with different concentrations of sodium lactate had no significant effect on the growth and development of zebrafish. The steps are as follows:

[0031] The experiment was conducted in an aquarium of 35cm×23.5cm×36cm, with water changed every day and the water temperature maintained at about 28°C. Adult AB zebrafish raised to 3 months old were mated and laid eggs, and the obtained eggs were placed in a 10cm culture dish and added with 10mL 0.1% methylene blue solution in a constant temperature incubator at 28°C. Dead eggs were picked out every 12 hours, and 30 zebrafish fry were picked out from each 6cm culture dish after incubation for 3 days as a treatment group, and a total of four treatment groups were set up.

[0032] Weigh 560.15 mg of sodium lactate and add 1 mL of water to prepare a sodium lactate stock solution with a final concentration of 5 M. Add the prepared sodium lactate stock solution to different treatment groups in amounts of 0 μL, 1 μL, 2 μL and 10 μL, and add 0.2% sea salt solution to a total volume of 5 mL. The final concentrations of sodium lactate in each group are 0 mM, 1 mM, 2 mM and 10 mM, respectively. Return each treatment group to a 28°C constant temperature incubator and continue incubating for 72 hours. Observe the growth and development of zebrafish fry under a microscope. The results are as follows: Figure 1 shown.

[0033] like Figure 1 As shown, observation under a microscope revealed that, similar to the control group of 0 mM sodium lactate, 1 mM, 2 mM, and 10 mM sodium lactate had no significant effect on the growth and development of zebrafish fry.

[0034] Example 2

[0035] Sodium lactate enhances the survival rate of zebrafish infected with SVCV, the steps are as follows:

[0036] AB type zebrafish were raised according to the method described in Example 1 and the adult fish were allowed to mate and lay eggs. The obtained eggs were placed in a 10 cm culture dish and 10 mL of 0.1% methylene blue solution was added and incubated in a constant temperature incubator at 28°C. Dead eggs were picked out every 12 h. Thirty zebrafish fry were picked out from each 6 cm culture dish after incubation for 3 days as a treatment group. A total of three experimental groups were set up.

[0037] Group A was the control group, and 5 mL of 0.2% sea salt solution was added; Group B was added with 2 mL of SVCV (titer was ~2.50×10 7 TCID 50 / mL) and supplemented with 3mL of 0.2% sea salt solution; Group C was infected with 2mL of SVCV (titer was ~2.50×10 7 TCID 50 / mL) and supplemented with 3mL of 0.2% sea salt solution and then added 5μL of 5M sodium lactate stock solution to ensure that the final concentration of sodium lactate was 5mM. Each treatment group was returned to the 28℃ constant temperature incubator for further incubation for 12h, and the growth and development of zebrafish fry were observed under a microscope. The results are as follows Figure 2 As shown, Figure 2 The red arrow in panel B indicates a curled-up zebrafish larva that died after being infected with SVCV.

[0038] like Figure 2 As shown in the figure, under the microscope, it was found that the mortality rate of zebrafish fry in group B was 30%, while after sodium lactate was added to group C, the mortality rate of zebrafish was 0. This shows that sodium lactate has a significant protective effect on zebrafish infected with SVCV.

[0039] Example 3

[0040] Sodium lactate inhibits the expression of virus-related genes in zebrafish infected with SVCV, the steps are as follows:

[0041] The control group and the experimental group were treated according to the method described in Example 2. After 12 hours of treatment, 30 zebrafish fry in each group were collected into a grinding tube and washed once with 0.2% sea salt solution. Total tissue RNA was extracted using TRIzol (Invitrogen), and cDNA was obtained by reverse transcription using the EasyScript reverse transcription kit (TRANS). The expression of virus-related genes was detected by RT-PCR, and the virus-related primers used were: the forward primer and reverse primer of SVCV-G-protein-RT were: 5'-CGACCTGGATTAGACT TG-3' (SEQ ID NO.1), 5'-AATGTTCCGTTTCTCACT-3' (SEQ ID NO.2);

[0042] The forward and reverse primers of SVCV-N-protein-RT were: 5′-TGAGGTGAGTGCT GAGGATG-3′ (SEQ ID NO. 3), 5′-CCATCAGCAAAGTCCGGTAT-3′ (SEQ ID NO. 4);

[0043] The forward primer and reverse primer of SVCV-P-protein-RT were: 5′-TTGGACCTGGGAT AGTGA-3′ (SEQ ID NO. 5), 5′-CTTGCTTGGTTTGTGGG-3′ (SEQ ID NO. 6);

[0044] The forward primer and reverse primer of SVCV-L-protein-RT were: 5′-GCCCACTTTGCATC CAGTCC-3′ (SEQ ID NO. 7), 5′-GAGATGCCACAGACTCCTCC-3′ (SEQ ID NO. 8);

[0045] The forward primer and reverse primer of SVCV-M-protein-RT were: 5'-TACTCCTCCCACT TACGA-3' (SEQ ID NO. 9), 5'-CAAGAGTCCGAGAAGGTC-3' (SEQ ID NO. 10), respectively.

[0046] The RT-PCR reaction system was: 28 μL enzyme-free water, 3.3 μL forward primer and reverse primer, 33 μL RT mix, 2 μL cDNA. Three replicates were set up with 20 μL of the mixture in each well, and the following program was run: 95°C for 5 min; 95°C for 3 s, 60°C for 15 s, for a total of 55 cycles; 65°C for 5 s; 95°C for 5 s.

[0047] Test results such as Figure 3 As shown. Figure 3 The results show that after treatment with 5 mM sodium lactate, the expression of virus-related genes SVCV-N, SVCV-M, SVCV-P, SVCV-G, and SVCV-L (corresponding to nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and RNA-dependent RNA polymerase (L)) in zebrafish larvae decreased significantly, indicating that sodium lactate has a significant inhibitory effect on the proliferation of SVCV in zebrafish.

[0048] Example 4

[0049] In this example, CRISPR / Cas9 technology was used to obtain zebrafish lacking lactate dehydrogenase (ldha), and SVCV was used to treat zebrafish lacking ldha and wild-type zebrafish. The specific process is as follows:

[0050] Knockout of ldha in zebrafish using CRISPR / Cas9:

[0051] First, sgRNA was designed using the CRISPR design tool (http: / / crispr.mit.edu), and the nucleotide sequence was 5'-GGAAGCTGAGCGGTTTGCCC-3' (SEQ ID NO.11). The optimized zebrafish codon Cas9 plasmid was obtained by digestion with Xba I, and the specific sequence was 5'-GTAATACGACTCACT ATAGGAAGCTGAGCGGTTTGCCCGTTTTAGAGCTAGAAATAGC-3' (SEQ ID NO.12), and then purified and transcribed. The ldhasgRNA template was amplified using the PpUC19-gRNA vector. sgRNA was synthesized using Transcript Aid T7 High Yield Transcription Kit (Fermentas). One-cell stage zebrafish embryos were injected with 1 ng Cas9 RNA and 0.15 ng sgRNA per embryo. Mutations were initially detected using heteroduplex mobility assay (HMA). If the HMA result was positive, the remaining embryos were cultured to adulthood as the F0 generation and then backcrossed with WT zebrafish (AB strain) to produce the F1 generation. The F1 generation was genotyped using HMA. The genotype was determined by target sequencing. The heterozygous F1 generation was backcrossed with WT zebrafish (AB strain) to produce the F2 generation. Heterozygous F2 generation adults carrying the target mutation were hybridized to produce the F3 generation. The ratio of wild type, heterozygous, and homozygous in the F3 generation should conform to Mendel's law of inheritance (1:2:1).

[0052] The ldha-deficient zebrafish (denoted as ldha) obtained in the above method were raised by the method described in Example 1. - / - ) and wild-type zebrafish (denoted as ldha + / + ) and the adult fish were mated and laid eggs. The obtained eggs were placed in a 10 cm culture dish and 10 mL of 0.1% methylene blue solution was added in a constant temperature incubator at 28°C. Dead eggs were picked out every 12 hours. A number of ldha-deficient zebrafish and wild-type zebrafish fry that had been incubated for 3 days were picked out and placed in a 6 cm culture dish.

[0053] ldha + / + The zebrafish larvae were divided into two groups. The first group was treated with 5 mL of 0.2% sea salt solution; the second group was treated with 2 mL of SVCV (titer of 2.50 × 10 7 TCID 50 / mL) infection and supplemented with 3mL 0.2% sea salt solution; ldha - / - Zebrafish larvae were also divided into two groups, with ldha + / + The zebrafish fry were treated in the same way. Each treatment group was placed back in the 28°C constant temperature incubator for another 12 hours, and the growth and development of the zebrafish fry were observed under a microscope. Figure 4As shown, Figure 4 The red arrow in the middle indicates a curled-up zebrafish larva that died after being infected with SVCV.

[0054] ldha + / + With ldha - / - Two groups of 96 zebrafish larvae were prepared. In the first group, each zebrafish larvae was carefully pipetted into a 96-well culture dish, and 100 μL of 0.2% sea salt solution was added to each well. In the second group, each zebrafish larvae was carefully pipetted into a 96-well culture dish, and then 20 μL of SVCV (titer of ∼2.50×10 7 TCID 50 / mL) and supplemented with 80 μL of 0.2% sea salt solution. Each treatment group was returned to the 28°C constant temperature incubator for further incubation. The death of zebrafish fry was counted under a microscope every hour. The results are as follows: Figure 4 shown.

[0055] ldha was treated according to the method in Example 3 + / + With ldha - / - RNA was extracted from zebrafish larvae and the expression of virus-related genes was detected by RT-PCR. The results are as follows Figure 4 shown.

[0056] like Figure 4 The results show that the mortality rate of ldha-deficient zebrafish infected with SVCV increased significantly. The replication of viral genes in zebrafish larvae was detected by RT-PCR (the method and primers were the same as in Example 3), and it was found that the expression levels of SVCV-N, SVCV-P, and SVCV-G in ldha-deficient zebrafish increased significantly.

[0057] It can be concluded from the above examples that lactate ions can inhibit the replication and proliferation of carp spring viremia virus, improve the survival rate of infected fish, and effectively prevent and treat carp spring viremia in the breeding process of carp fish.

[0058] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of sodium lactate as an active ingredient in the preparation of products for preventing and treating carp spring viremia; The lactate ion is used at a concentration of 5-10 mM.

2. The use according to claim 1, characterized in that: The product for preventing and treating carp spring viremia includes one or more products of enhancing the fish body's resistance to carp spring viremia virus infection, inhibiting the replication of carp spring viremia virus infection and inhibiting the proliferation of carp spring viremia virus infection.

3. The use according to claim 1, characterized in that: The product includes a medicine or a feed.

4. The use according to claim 3, characterized in that: The dosage form of the drug includes liquid or solid.

5. The use according to claim 4, characterized in that: The liquid preparation includes an injection or an immersion preparation.

6. The use according to claim 4, characterized in that: The feed comprises aquatic feed and a lactate ion component.

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