Bacillus velezensis with antiviral and detoxification functions and application thereof

Abstract

The application discloses a bacillus velezensis with antiviral and detoxification functions and an application thereof, relates to the field of animal antifungal and viral microbiology technology, and the bacillus velezensis is preserved in the China Center for Type Culture Collection on June 8, 2018, and the preservation number is CCTCC NO: M2018352. The bacillus velezensis in the application plays an intervention role in the combined infection of mycotoxin DON and virus PCV2, can significantly inhibit the infection of the virus, helps the body to restore the immune response ability, removes the inhibition of DON on the cGAS-STING antiviral signal path mediated by type I interferon, and the intervention role of the bacillus velezensis on the combined infection of DON and PCV2 is achieved by inhibiting the occurrence of cell autophagy. The bacillus velezensis can significantly inhibit the proliferation of PCV2 and the promoting effect of DON on the proliferation of PCV2, and the bacillus velezensis has a good application prospect in products such as probiotic preparations or feed additives.

Description

Technical Field

[0001] This invention relates to the field of animal antifungal and viral microbiology technology, specifically to a strain of Bacillus bellis with antiviral and detoxification functions and its applications. Background Technology

[0002] Deoxynivalenol (DON), a type of trichothecene fungal toxin, can enter the food chain through grains and feed, posing a threat to animal and human health. The cytotoxicity and immunotoxicity of DON can exacerbate infections caused by other pathogens. Pigs are particularly susceptible to DON, and its toxic effects have a significant impact on swine herds. Porcine circovirus type 2 (PCV2) is a common disease in pig farms, causing porcine circovirus-related diseases, leading to loss of appetite, emaciation, immune dysfunction, and even death in severe cases.

[0003] Bacillus belye was isolated in 1999 and first reported and named in 2005. It is an aerobic, Gram-positive bacillus with endospores, widely distributed in nature in water, soil, air, plant roots, plant surfaces, and animal intestines. Due to its role in promoting plant and animal growth, resisting pathogens, and enhancing the body's immune response, it plays an important role in biological control, drug development, food fermentation, and industrial applications.

[0004] Several studies have reported that *Bacillus belycera* can improve inflammatory damage caused by toxins such as zearalenone and deoxynivalenol. However, research on co-infection with DON and PCV2 has not yet been reported. In actual production, pig farms infected with PCV2 experience a sharp increase in morbidity and mortality after DON contamination. Therefore, we urgently need to find a probiotic that can resist co-infection with DON and PCV2. This application is the first to discover a *Bacillus* strain resistant to co-infection with PCV2 and DON, and in vitro studies have shown that it can resist co-infection and reduce PCV2 proliferation in cells, providing a new product for the prevention and control of the harm caused by co-infection with PCV2 and DON in actual production. Summary of the Invention

[0005] The purpose of this invention is to provide a strain of Bacillus belye with antiviral and detoxification functions and its application. This strain is derived from soil and has the ability to effectively resist infection by fungal toxins and pathogenic microorganisms, thereby solving the economic losses caused by mixed infection of DON and PCV2 in pig farms.

[0006] To achieve the above objectives, this invention analyzed the role of Bacillus belyss in the inflammatory damage of porcine kidney cells induced by the co-infection of DON and PCV2, and subsequently found that Bacillus belyss has an interventional role in the promotion of PCV2 infection by DON.

[0007] Based on the above findings, this study explored the specific mechanism by which Bacillus belysin intervenes with DON to promote PCV2 infection. It was found that DON can inhibit the activation of the antiviral signaling pathway cGAS-STING, while Bacillus belysin can relieve the inhibitory effect of DON, thereby relieving the expression of type I interferon and antiviral proteins inhibited by DON, thus resisting the invasion of pathogenic microorganisms and fungal toxins.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] A strain of Bacillus velezensis with antiviral and detoxification functions was deposited at the China Center for Type Culture Collection on June 8, 2018, with accession number CCTCC NO:M2018352.

[0010] Application of a strain of Bacillus vesiculosus with antiviral and detoxification functions, and its use as an active ingredient in probiotics.

[0011] As a further aspect of the present invention: the use of the aforementioned Bacillus berberis as a therapeutic agent for diseases caused by porcine circovirus and deoxynivalenol.

[0012] As a further aspect of the present invention: the application of the aforementioned Bacillus amyloliquefaciens in agricultural production for the removal of mycotoxin DON contamination.

[0013] Compared with the prior art, the beneficial effects of the present invention are:

[0014] 1. Bacillus belyss plays an interventional role in the co-infection of mycotoxin DON and virus PCV2. It can significantly inhibit viral infection, help the body restore its immune response, and relieve the inhibitory effect of DON on the cGAS-STING antiviral signaling pathway mediated by type I interferon.

[0015] 2. The intervention effect of Bacillus belye against DON and PCV2 co-infection is achieved by inhibiting autophagy.

[0016] 3. This invention proposes the application of Bacillus belye: to be made into feed additives or medicinal food products to prevent the occurrence of diseases caused by the combined infection of mycotoxin DON and porcine circovirus. Attached Figure Description

[0017] Figure 1 This is a screening diagram showing the effective intervention concentration and optimal treatment time of Bacillus vesiculosus in this invention.

[0018] Figure 2This diagram illustrates how Bacillus belye, as described in this invention, relieves the upregulation of PCV2 proliferation by DON through autophagy.

[0019] Figure 3 This diagram illustrates the effects of Bacillus vesiculosus of the present invention on type I interferon and antiviral proteins OAS, MX1, and the immune cytokine IL-10.

[0020] Figure 4 The diagram illustrates the effect of Bacillus belysus in relieving the inhibition of the cGAS-STING pathway by DON. Detailed Implementation

[0021] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] Figure 1 This study aimed to screen for effective intervention concentrations and optimal treatment times for *Bacillus belyssus*. All data are expressed as mean ± standard deviation. Compared to the control group, * P < 0.05 indicates a significant difference. ** P < 0.01 indicates that the difference is highly significant.

[0023] (A) The effective intervention concentration of different concentrations of Bacillus belyssus after co-infection with PCV2 and DON was determined by the MTT method;

[0024] (B) Optimal treatment time for Bacillus belesi was determined by the MTT assay.

[0025] (C) The expression of LC3B protein under different pretreatment times of Bacillus belysaeus was detected by Western blot.

[0026] Figure 2 This study investigated how Bacillus belye relieved the upregulation of PCV2 proliferation by DON through autophagy. All data are expressed as mean ± standard deviation. Compared with the control group, * P < 0.05 indicates a significant difference. ** P < 0.01 indicates a highly significant difference; compared with the DON group, # P < 0.05 indicates a significant difference. ## P < 0.01 indicates that the difference is highly significant.

[0027] (A) The relative expression level of PCV2 capsid protein CAP in each treatment group after Bacillus belysin pretreatment was detected by Western blot.

[0028] (B) The relative expression level of LC3B, the main autophagy protein, in each treatment group after Bacillus belysin pretreatment was detected by Western blot.

[0029] (C) The relative expression level of LC3B mRNA, the main autophagy protein, in each treatment group after Bacillus bereavement pretreatment was detected by RT-qPCR.

[0030] Figure 3 This study investigated the effects of Bacillus belyssus on type I interferon and antiviral proteins OAS and MX1, as well as the immune cytokine IL-10. Data are presented as mean ± standard deviation. Compared with the control group, * P < 0.05 indicates a significant difference. ** P < 0.01 indicates a highly significant difference; compared with the DON group, # P < 0.05 indicates a significant difference. ## P < 0.01 indicates that the difference is highly significant.

[0031] (A) The relative expression levels of IFN-α and IFN-β mRNA in each treatment group after pretreatment with Bacillus belysinus were detected by RT-qPCR.

[0032] (B) The relative expression levels of OAS and MX1 mRNA in each treatment group after Bacillus belysin pretreatment were detected by RT-qPCR.

[0033] (C) The relative expression level of IL-10 mRNA in each treatment group after Bacillus belysin pretreatment was detected by RT-qPCR.

[0034] Figure 4 To relieve the inhibitory effect of DON on the cGAS-STING pathway by Bacillus belysinus. All data are expressed as mean ± standard deviation. Compared with the control group, * P < 0.05 indicates a significant difference. ** P < 0.01 indicates a highly significant difference; compared with the DON group, # P < 0.05 indicates a significant difference. ## P < 0.01 indicates that the difference is highly significant.

[0035] The relative expression levels of cGAS-STING signaling pathway-related proteins (cGAS, STING, P-STING, IRF3, P-IRF3, β-actin) in each treatment group after Bacillus belyi pretreatment were detected by Western blot.

[0036] Example 1

[0037] A strain of Bacillus velezensis with antiviral and detoxification functions and its application. This Bacillus velezensis was deposited at the China Center for Type Culture Collection on June 8, 2018, with accession number CCTCC NO:M2018352, at Wuhan University, Wuhan, China.

[0038] Example 2

[0039] Screening of effective intervention concentrations and optimal treatment times for Bacillus belyssus.

[0040] To ensure that the added *Bacillus belyssus* effectively intervenes in the co-infection of PCV2 and DON without causing cell damage, the effective intervention concentration of *Bacillus belyssus* was first investigated, such as... Figure 1 As shown in Figure A, compared with the control group (DON), except for 2.1 × 10 8 Except for the cfu / mL group, which showed a highly significant decrease in cell viability after DON upregulation of PCV2 infection, all other groups were able to alleviate the damage caused by combined infection to cells. The 2.1×106 cfu / mL group significantly alleviated the damage caused to cells after DON upregulation of PCV2 infection.

[0041] To determine the optimal treatment time for Bacillus belyssus, this invention employs the MTT assay and Western blotting for detection, such as... Figure 1 As shown in B / C, compared with the control group, Bacillus belysin pretreatment for 2h, 6h and 12h significantly reduced the expression level of LC3B protein and significantly alleviated the damage caused by DON to cells. Moreover, the decrease in LC3B protein expression was more significant when Bacillus belysin pretreatment was 12h, and its intervention effect was better. At the same time, it is speculated that Bacillus belysin can alleviate the infection caused by DON upregulation of PCV2 by inhibiting autophagy.

[0042] In summary, a Bacillus belyss concentration of 2.1 × 10⁶ cfu / mL and a pretreatment time of 12 h were selected as the subsequent treatment conditions.

[0043] Example 3

[0044] Bacillus belye can reverse the upregulation of PCV2 proliferation by DON through autophagy.

[0045] To investigate the intervention effect of Bacillus belycetamol on DON-promoted PCV2 infection, this invention uses Western blotting to detect the major structural protein CAP of PCV2, such as... Figure 2As shown in Figure A, after treatment with DON in PCV2-infected cells, the expression level of CAP protein significantly increased, while the expression level of CAP protein significantly decreased in the *Bacillus belyssum* single group and the *Bacillus belyssum* pretreatment group. Subsequently, the study investigated whether this intervention effect of *Bacillus belyssum* was related to autophagy. Western blotting and quantitative real-time PCR were used to detect the protein and gene expression of LC3B, a major autophagy protein. Figure 2 As shown in B / C, compared with the control group, the expression levels of LC3 protein and mRNA were significantly increased in the single DON group, while the Bacillus belyss pretreatment group blocked this effect of DON.

[0046] In summary, Bacillus belye can alleviate the effect of DON upregulating PCV2 infection by inhibiting autophagy.

[0047] Example 4

[0048] Effects of Bacillus vesiculosus on type I interferon, antiviral proteins, and immune cytokines.

[0049] To investigate how the intervention effect of Bacillus belyssus occurs, this invention uses RT-qPCR to detect type I interferons (IFN-α and IFN-β), antiviral proteins (OAS and MX1), and the immune cytokine IL-10 mRNA, such as... Figure 3 In groups A / B / C, the expression levels of IFN-α, IFN-β, OAS, and MX1 mRNA were significantly decreased in the DON treatment group, while the expression level of IL-10 mRNA was significantly increased. However, the addition of Bacillus belye pretreatment significantly blocked the effects of DON, resulting in a significant increase in the expression levels of IFN-α, IFN-β, OAS, and MX1 mRNA, while the expression level of IL-10 mRNA was significantly decreased.

[0050] In summary, Bacillus belyssus can significantly increase the expression of type I interferon and antiviral proteins, and can alleviate the immunosuppressive effect caused by DON upregulation of PCV2 infection.

[0051] Example 5

[0052] Bacillus belye relieves the inhibitory effect of DON on the cGAS-STING pathway.

[0053] The cGAS-STING signaling pathway is an important antiviral signaling pathway. It regulates the production of antiviral proteins by modulating the expression of type I interferon, thereby exerting its effect. To investigate whether the intervention effect of Bacillus bereaves is related to the cGAS-STING signaling pathway, this invention uses Western blotting to detect the expression of pathway proteins and downstream proteins, such as... Figure 4 As shown, compared with the control group, the expression levels of cGAS, phosphorylated STING, and phosphorylated IRF3 were significantly increased in the Bacillus belysium single-done group, while they were significantly decreased in the DON single-done group. Compared with the DON single-done group, the expression levels of cGAS, phosphorylated STING, and phosphorylated IRF3 were significantly increased in the Bacillus belysium pretreatment group. Meanwhile, the expression of total STING and IRF3 proteins remained unchanged during the experiment.

[0054] In summary, Bacillus belye can relieve the inhibitory effect of DON on the cGAS-STING signaling pathway, thereby enabling it to exert its antiviral effect.

[0055] This application provides a strain of Bacillus belyssus and its application in inhibiting the co-infection of PCV2 and DON. Thanks to the ability of Bacillus belyssus to significantly inhibit the proliferation of PCV2 and the promoting effect of DON on the proliferation of PCV2, it has good application prospects in products such as probiotic preparations or feed additives.

[0056] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of *Bacillus belyssus* in the preparation of drugs for intervening in diseases caused by co-infection of porcine circovirus type 2 and deoxynivalenol, characterized in that, The Bacillus belesi ( Bacillus velezensis The *Bacillus belyssus* strain was deposited at the China Center for Type Culture Collection on June 8, 2018, with accession number CCTCC NO: M2018352. It inhibits the proliferation of porcine circovirus type 2 by suppressing autophagy, thereby relieving the inhibitory effect of deoxynivalenol on the type I interferon-mediated cGAS-STING antiviral signaling pathway.

2. The application according to claim 1, characterized in that, The drug is a probiotic preparation.

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