ZSJB6 and application thereof

Abstract

This invention relates to the field of microbial technology, specifically to a strain of Veillonella ZSJB6 and its applications. The Veillonella strain provided by this invention... Veillonella sp. ZSJB6 is a new species of Veillonella. Its culture supernatant has a good inhibitory effect on VSV virus replication. Hemolysis and cytotoxicity experiments show that this strain meets the safety requirements and can provide a new means for the prevention and control of VSV infection.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a strain of Veillonella ZSJB6 and its applications. Background Technology

[0002] The information disclosed in the background section of this invention is intended only to enhance the understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Veillonella spp. ( Veillonella *Bacillus* is a group of Gram-negative, strictly anaerobic bacteria, 0.3–0.5 μm in diameter, appearing as diplococci, plates, and short chains under a microscope. They lack capsules, flagella, and spores, and are widely colonized in the human oral cavity and intestines. This genus currently contains 15 known species, namely… Veillonella a ty pica , Veillonella ca via e , Veillonella hamsters , Veillonella denticaria , Veillonella dispar , Veillonella infant , Veillonella magna , Veillonella montpellierensis , Veillonella nakazawae , Veillonella parvula , Veillonella ratti , Veillonella rodentium , Veillonella rogosae , Veillonella seminalis , Veillonella tobetsuensis In the oral and intestinal environment, Veillonella cocci maintain the balance of the gut microbiota through metabolic interactions, biofilm formation, and immune regulation, in conjunction with other microorganisms.

[0004] Vesicular stomatitis virus (VSV) is an RNA virus with broad host adaptability and zoonotic characteristics. This virus can infect various livestock (such as cattle, horses, and pigs), wild animals (including rodents), birds, and fish, and can also cause infection in humans. Infected animals often exhibit typical oral and hoof vesicular lesions, while humans often experience flu-like symptoms such as pain, fever, and sore throat after contact. Due to the wide transmission range, high infectivity, and variability of VSV, a safe and effective control mechanism has not yet been established, and its epidemic and control remain significant challenges for veterinary and public health fields. Summary of the Invention

[0005] In view of this, the present invention provides a strain of Veillonella ZSJB6 and its applications.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0007] In a first aspect, the present invention provides a strain of Veillonella cocci. Veillonella sp. ZSJB6 was deposited on August 4, 2025, at the China Center for Type Culture Collection (CCTCC), located in Wuhan, China, with accession number CCTCC NO: M20251752.

[0008] Secondly, the present invention provides a microbial inoculant containing the aforementioned Veillonella cocci. Veillonella sp. ZSJB6 or its ferments or metabolites.

[0009] In this invention, the term "fermentation product" is used to refer to fermentation products. The corresponding fermentation product can be derived from the fermentation culture of *Veillonella*. Veillonella sp. The liquid obtained from the ZSJB6 process can therefore also be called fermentation broth; the liquid may contain bacteria (bacterial cells), but it is not necessarily required to contain bacteria. The liquid preferably contains *Veillonella* of the present invention. Veillonella sp. Metabolites produced by ZSJB6.

[0010] Furthermore, in embodiments of the present invention, the fermentation broth or culture medium containing bacterial cells is separated from the liquid by centrifugation, filtration, sedimentation, or other means known in the art. The liquid remaining after removing the bacterial cells is called the "supernatant." In the present invention, the supernatant contains Veillonella cocci. Veillonella sp. The extracellular metabolites of ZSJB6. In embodiments of the present invention, the bacterial agent may also contain this supernatant.

[0011] In summary, among the aforementioned microbial agents, the above-mentioned Veillonella cocci... Veillonella sp. ZSJB6 is alive or dead or intermittently sterilized, or in the form of lysates and / or extracts, or in the form of bacterial products, supernatants, or derivatives, said derivatives including: metabolites, metabolobioproducts, probiotics, extracellular polysaccharides, and compounds containing immunogenic components.

[0012] Preferably, the Veillonella cocci Veillonella sp. ZSJB6 is a live bacterium, an inactivated bacterium, or a bacterium that has been attenuated.

[0013] Preferably, the microbial agent is an inactivated agent or a live agent.

[0014] Preferably, the microbial agent is a liquid agent or a solid agent.

[0015] Thirdly, the present invention provides the *Veillonella* strain described in the first aspect. Veillonella sp.Application of ZSJB6 or the microbial agent described in the second aspect in the preparation of products for treating VSV virus infection-related diseases.

[0016] Preferably, the VSV virus infection-related disease is vesicular stomatitis.

[0017] Preferably, the product is a drug.

[0018] Preferably, the drug comprises pharmaceutically acceptable excipients and / or adjuvants.

[0019] Preferably, the Veillonella cocci Veillonella sp. ZSJB6 is the only active ingredient in the drug.

[0020] Preferably, the drug is an oral preparation or an injection; the oral preparation is a powder, granule, tablet, capsule, suspension, emulsion, syrup or spray.

[0021] Preferably, the excipients and / or additives include nutrient fortifiers, sweetness regulators, acidity regulators, isotonic regulators, fillers, binders, disintegrants, solubilizers, cosolvents, preservatives, flavoring agents, colorants, suspending agents, wetting agents, emulsifiers and / or surfactants.

[0022] Preferably, the drug may further comprise a suitable amount of commonly used carrier, excipient, and diluent. Moreover, it can be formulated into oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and sprays, as well as sterile injectable solutions, according to conventional methods. The non-pharmaceutical active ingredients such as carriers, excipients, and diluents that can be included in this invention are well known in the art, and those skilled in the art can determine that they meet clinical standards. The carriers, excipients, and diluents of this invention include, but are not limited to, lactose, glucose, sorbitol, mannitol, xylitol, maltitol, starch, gum arabic, gelatin, calcium phosphate, cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, and mineral oil. No specific limitations are made herein.

[0023] Fourthly, the present invention provides a medicine comprising the Veillonella cocci described in the first aspect. Veillonella sp. ZSJB6 or the microbial agent described in the second aspect.

[0024] Preferably, the drug is a drug for the prevention, improvement or treatment of diseases related to VSV virus infection.

[0025] Compared with the prior art, the present invention has achieved the following beneficial effects:

[0026] (1) Veillonella provided by the present invention Veillonella sp.ZSJB6 is a new species of Veillonella, and its culture supernatant has a good inhibitory effect on VSV virus replication, which can provide a new means for the prevention and control of VSV infection.

[0027] (2) Veillonella provided by the present invention Veillonella sp. ZSJB6 was isolated from healthy human samples, showing no species rejection reaction. Hemolysis and cytotoxicity tests indicated that it was *Veillonella*. Veillonella sp. ZSJB6 meets security requirements and is valuable for application transformation. Attached Figure Description

[0028] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0029] Figure 1 Veillonella Veillonella sp. Scanning electron microscope image of ZSJB6;

[0030] Figure 2 Veillonella Veillonella sp. ZSJB6 is based on a genome-wide multiple sequence typing phylogenetic tree;

[0031] Figure 3 Veillonella Veillonella sp. Figure showing the results of the hemolytic activity test of ZSJB6;

[0032] Figure 4 Veillonella Veillonella sp. Figure showing the cytotoxicity results of ZSJB6 metabolites;

[0033] Figure 5 Veillonella Veillonella sp. Figure 1. Fluorescence microscopy results of ZSJB6 supernatant inhibiting VSV replication;

[0034] Figure 6 Veillonella Veillonella sp. The qPCR detection results of ZSJB6 supernatant inhibiting VSV replication are shown in the figure; (A) is the qPCR detection result in INT407 cells, and (B) is the qPCR detection result in THP-1 cells. Detailed Implementation

[0035] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0036] The culture medium used in the examples has the following composition:

[0037] The composition of BHI liquid culture medium is as follows:

[0038] 10 g peptone, 12.5 g dehydrated calf brain extract powder, 5 g dehydrated calf heart extract powder, 5 g sodium chloride, 2 g glucose, 2.5 g disodium hydrogen phosphate, and 1 mg resazurin were dissolved in 1 L of water and the pH was adjusted to 7.4 ± 0.2.

[0039] DMEM culture medium uses commercially available finished culture medium, brand code as follows: Gibco, C11995500BT.

[0040] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0041] Example 1: A strain of Veillonella cocci Veillonella sp. Isolation, purification and identification of ZSJB6

[0042] (1) Isolation and purification of Veillonella ZSJB6: Fecal samples were collected from healthy adults, diluted with physiological saline, and spread onto brain and heart infusion agar plates. The plates were then anaerobically cultured at 37°C for 2-3 days. Single colonies were picked from the plates and streaked for purification. The purified colonies were identified using MALDI-TOF MS and 16S rDNA full-length sequencing. The preliminary identification result for this strain was Veillonella.

[0043] (2) Morphological observation of Veillonella ZSJB6: ZSJB6 strain was inoculated into BHI liquid medium and cultured anaerobicly at 37°C for 20 h. The cells were then collected by centrifugation at 5000 rpm for 5 min. The cells were gently washed twice with sterile phosphate-buffered saline (PBS), resuspended in 2.5% glutaraldehyde, and fixed overnight at 4°C. After fixation, the cells were collected by centrifugation at 8000 rpm for 1 min, the supernatant was discarded, and the cells were washed three times with PBS buffer. The cells were then dehydrated in a gradient of 30%, 50%, 70%, 90%, and 100% ethanol, and finally resuspended in anhydrous ethanol. 10 μL of the bacterial suspension was dropped onto a glass slide, dried, and sputter-coated with gold. The morphology of the cells was observed using a scanning electron microscope. Figure 1 Veillonella Veillonella sp. Scanning electron microscope morphology image of ZSJB6, as shown Figure 1 As shown, ZSJB6 cells are similar to existing Veillonella cells. ZSJB6 cells are spherical, with a diameter of about 0.3-0.5 μm, and lack flagella.

[0044] (3) Whole genome sequencing analysis of Veillonella ZSJB6: The whole genome of the strain was sequenced using the Illumina Miseq sequencing platform, and the data was assembled using SPAdes (v3.15.5) software. The genome data has been uploaded to the NCBI (National Center of Biotechnology Information) public platform, sequence number JBPUGE000000000. Based on the whole genome sequencing results, tandem phylogenetic tree analysis was performed on housekeeping gene sequences such as ribosomal protein coding genes (rplV, rplN, rplP), DNA helicase gene dnaB, and RNA polymerase subunit gene rpoA. Figure 2 Veillonella Veillonella sp. ZSJB6 is based on a genome-wide multiple sequence typing phylogenetic tree. For example... Figure 2 As shown, strain ZSJB6 and *Veillonella spp.* (…) Veillonella dispar The strains clustered together, exhibiting the highest similarity. Further calculations were made of the average nucleotide identity (ANI) of *Veillonella* ZSJB6 with other closely related strains, and homology between strains was calculated using digital simulated DNA-DNA hybridization (dDDH). The results are shown in Table 1. Strain ZSJB6 showed the highest ANI value (95.9%) and dDDH value (55.8%) among closely related strains. According to existing species classification standards, an ANI > 96% and a dDDH > 60% are required to be considered the same species. Therefore, ZSJB6 was identified as a novel *Veillonella* species.

[0045] Table 1. Analysis of ANI and dDDH in Veillonella ZSJB6

[0046]

[0047] Example 2: Virulence test of Veillonella ZSJB6

[0048] (1) Hemolytic test of Veillonella ZSJB6: Veillonella ZSJB6 was inoculated into BHI liquid medium and cultured overnight at 37°C. The activated bacterial solution was taken with an inoculation loop and inoculated onto Columbia blood agar plates using the three-zone streak method. After anaerobic culture at 37°C for 24 h, the presence of hemolysis was observed. Figure 3 Veillonella Veillonella sp. The results of the ZSJB6 hemolytic test are shown in the figure. Figure 3 As shown, no hemolytic zone was observed around the colony, indicating that the strain does not produce hemolysin and therefore poses no corresponding toxicity risk.

[0049] (2) Cytotoxicity detection of Veillonella ZSJB6 metabolites: ZSJB6 strain was inoculated into BHI liquid medium and cultured overnight at 37°C. The supernatant was collected by centrifugation at 7000 rpm for 5 min and filtered through a 0.22 μm filter membrane. Human small intestinal epithelial cells INT407 were seeded into 96-well plates at a density of 1×10⁻⁶. 4 / well, incubated at 37℃. After cell attachment, different doses of ZSJB6 bacterial supernatant were added (0.4 μL, 0.8 μL, 1.6 μL, 3.2 μL, 6.25 μL, 12.5 μL, 25 μL, 50 μL), with blank BHI liquid medium as a control. After co-incubation for 24 h, the supernatant was removed, and fresh DMEM medium and CCK8 reagent were added. The absorbance was measured at 450 nm. Figure 4 Veillonella Veillonella sp. The cytotoxicity results of ZSJB6 metabolites are shown in the figure. Figure 4 As shown, different doses of bacterial supernatant did not significantly affect the viability of INT407 cells, indicating that Veillonella ZSJB6 metabolites do not possess cytotoxicity.

[0050] Example 3: In vitro experiment on the inhibition of VSV replication by supernatant of Veillonella ZSJB6 bacterial culture

[0051] (1) Observation of green fluorescent protein-labeled VSV (VSV-GFP) infection by fluorescence microscopy: INT407 cells were seeded into 12-well plates (seedling density of 1×10⁻⁶ cells / wells). 5 After culturing for 12 h, 20 μL of Veillonella ZSJB6 bacterial supernatant or an equal volume of BHI blank medium was added, followed by inoculation with VSV-GFP (MOI = 0.1). Cells were collected 24 h after infection. The morphology and infection status of INT407 cells were observed and photographed under bright field and fluorescence conditions using a fluorescence microscope. Figure 5 Veillonella Veillonella sp. The image shows the fluorescence microscopy results of the ZSJB6 supernatant inhibiting VSV replication. (See figure.) Figure 5 As shown, compared with the control group with BHI blank medium, the addition of ZSJB6 bacterial supernatant can significantly inhibit the number of VSV-GFP infected cells (reduced green fluorescence intensity), and at the same time, it also improves the cytopathic effect caused by viral infection (cell morphology becomes round and detaches).

[0052] (2) qPCR detection of VSV mRNA levels: INT407 cells and THP-1 cells were seeded into 12-well plates (seedling density of 1×10⁻⁶ cells / well). 5 / well), after culturing for 12 h, 20 μL of Veillonella ZSJB6 bacterial supernatant or an equal volume of BHI blank medium was added, followed by inoculation with VSV-GFP (MOI = 0.1). INT407 and THP-1 cells infected with VSV-GFP for 24 h were collected, and total RNA was extracted using Trizol reagent. The RNA was then reverse transcribed into cDNA using a reverse transcription kit. The cDNA was diluted 50-fold, and the expression of VSV viral glycoprotein (GP) was detected by qPCR. ACTB was selected as the internal reference gene. Primer sequences are detailed in Table 2. Figure 6 Veillonella Veillonella sp. The image shows the qPCR detection results of ZSJB6 supernatant inhibiting VSV replication. Figure 6 In the diagram, (A) shows the qPCR detection results in INT407 cells, and (B) shows the qPCR detection results in THP-1 cells. Figure 6 As shown, treatment with the supernatant of Veillonella ZSJB6 bacterial culture significantly reduced the mRNA level of VSV in cells, with inhibition rates of 99% and 60% in INT407 and THP-1 cells, respectively, indicating that Veillonella ZSJB6 metabolites have a good inhibitory effect on VSV virus replication.

[0053] Table 2 Primer Sequences

[0054]

[0055] In Table 2, F is the forward primer (5'→3') and R is the reverse primer (5'→3').

[0056] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A strain of Veillonella ( Veillonella sp. ZSJB6, characterized in that, It was deposited on August 4, 2025 at the China Center for Type Culture Collection, located in Wuhan, China, with accession number CCTCC NO: M 20251752.

2. A microbial inoculant, characterized in that, Contains the Veillonella as described in claim 1 ( Veillonella sp. ZSJB6 or its fermentation supernatant.

3. The microbial agent as described in claim 2, characterized in that, The microbial agent is a solid agent.

4. The microbial agent as described in claim 2, characterized in that, The microbial agent is a live bacteria agent.

5. The Veillonella as described in claim 1 ( Veillonella sp. The application of the microbial agent described in ZSJB6 or claim 2 in the preparation of products for treating VSV virus infection-related diseases.

6. The application as described in claim 5, characterized in that, The product is a drug, which contains pharmaceutically acceptable excipients; the VSV virus infection-related disease is vesicular stomatitis.

7. The application as described in claim 6, characterized in that, The Veillonella ( Veillonella sp. ZSJB6 is the only active ingredient in the drug.

8. The application as described in claim 6, characterized in that, The drug is an oral or injectable preparation; the oral preparation is an oral preparation of powder, granules, tablets, capsules, suspension, emulsion, syrup or spray.

9. A drug, characterized in that, Contains the Veillonella as described in claim 1 ( Veillonella sp. ZSJB6.

10. The medicament as claimed in claim 9, characterized in that, The drug is a medicine for the prevention, improvement or treatment of diseases related to VSV virus infection.

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