Application of spirillum camarum CLA-AA-M04 in resisting pseudorabies virus infection

Abstract

The invention belongs to the field of agricultural microbe application, and particularly relates to an application of spirillum camarum CLA-AA-M04 in resisting pseudorabies virus infection. The trichospirillum is an anaerobic neutrophile bacterium and has a gram positive ultrastructure. The verification shows that the strain plays an antiviral role through the mechanisms of improving the respiratory tract barrier function, inhibiting virus replication, reducing inflammatory response and the like. By drenching the helispirillum caffei CLA-AA-M04, the virus load of the lung and brain of the PRV infected mouse can be obviously reduced, the survival rate is improved, the pathological injury of the lung is relieved, and the expression of inflammatory factors TNF-alpha and IL-6 is inhibited. In addition, the bacterial strain repairs the epithelial barrier function of the respiratory tract by up-regulating the expression of tight connection molecules, ZO-1 and Occludin and the expression of adhesion connection molecules E-cadherin and beta-catenin. The strain can be applied to preparation of a microbial preparation for resisting pseudorabies virus infection.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial medicine, and in particular to application of Lachnospira CLA-AA-M04 in resisting pseudorabies virus (PRV) infection. Background Art

[0002] Pseudorabies virus (PRV) is an acute infectious disease caused by PRV that can infect a variety of mammals, including pigs and cattle, causing respiratory damage, neurological symptoms, and high mortality. Vaccines are an ideal means of preventing and controlling viral transmission, but viral evolution may lead to immune escape. Cases of PRV infection leading to severe respiratory symptoms and even death in fattening pigs have been observed in clinical practice, suggesting limitations of traditional vaccine prevention and control strategies. In recent years, breakthroughs have been made in the study of the complex interactions between hosts and microorganisms. Probiotics have become a new direction in antiviral research due to their high safety, immune regulation, and barrier function.

[0003] Lachnospiraceae bacterium is an anaerobic neutrophil with a Gram-positive ultrastructure that inhabits a variety of ecosystems, including the gastrointestinal tracts of humans, mice, insects, and ruminants. A growing number of studies have identified Lachnospiraceae as an emerging biotherapeutic agent, effective in alleviating intestinal infections, inflammatory and allergic diseases, and even cancer, but its application in the antiviral field has not been reported. This study first discovered that Lachnospiraceae CLA-AA-M04 can inhibit PRV infection through multiple pathways, providing a new technical solution for PRV prevention and control. Summary of the Invention

[0004] The present invention provides use of Lachnospira CLA-AA-M04 and / or its culture in preparing a product for preventing and treating PRV infection.

[0005] The Lachnospiraceae bacterium CLA-AA-M04 used in the present invention was purchased from Mingzhou Biotechnology in Ningbo, Zhejiang, China. This Lachnospiraceae bacterium is classified as Lachnospiraceae bacterium. The applicant deposited this strain with the China Center for Type Culture Collection, Wuhan University, Wuhan, China on June 17, 2025, with the deposit number CCTCC M20251413.

[0006] The applicant used the strain Lachnospiraceae CLA-AA-M04 to conduct experiments and functional verification on mice and found that the strain can significantly reduce the viral load of the lung tissue and brain tissue of mice infected with pseudorabies virus (PRV) (see Figure 1 ), and can also improve the survival rate of experimental mice (see Figure 2 Histological analysis showed that the strain could reduce pathological damage such as alveolar wall thickening and inflammatory infiltration in mice (see Figure 3 ) and inhibit the expression of TNF-α and IL-6 (see Figure 4 In addition, Lachnospira CLA-AA-M04 can upregulate the expression of barrier proteins such as ZO-1 and Occludin (see Figure 5 ) and can repair the permeability of the mouse respiratory epithelial barrier (see Figure 6 ).

[0007] The new use of this strain is that it can be applied to the imitation of porcine pseudorabies virus, such as the preparation of related microbial preparations.

[0008] Preferably, the product dosage form includes capsules, tablets or oral liquid, and the administration route is oral or aerosol inhalation.

[0009] For a more detailed solution, please refer to the specific implementation method. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 : Comparison of viral load between Lachnospira intervention group and PRV infection group.

[0011] Figure 1 Figure A shows the results of the viral load test in the brain tissue of PRV-infected mice treated with Lachnospira.

[0012] Figure 1 Figure B is the result of viral load detection in lung tissue of mice infected with PRV with Lachnospira intervention.

[0013] Figure 2 : Changes in the survival rate of mice in each experimental group infected with PRV after intervention by Lachnospira.

[0014] Figure 3 :HE staining of lung tissue pathological sections of mice infected with PRV after Lachnospira intervention.

[0015] Figure 3 Panel A is the PBS mock control group;

[0016] Figure 3 Panel B in the figure is the PRV-infected group;

[0017] Figure 3 Figure C is the Lachnospira intervention PRV infection group.

[0018] Figure 4 : Effects of Lachnospira intervention on serum inflammatory factor levels in PRV-infected mice.

[0019] Figure 4 Figure A shows the level of inflammatory factor TNF-α in the serum of PRV-infected mice treated with Lachnospira;

[0020] Figure 4 Figure B shows the effect of Lachnospira on the level of inflammatory factor IL-6 in the serum of PRV-infected mice.

[0021] Figure 5 : Quantification of Evans blue in the lungs of PRV-infected mice after Lachnospira intervention.

[0022] Figure 5 Panel A is the PBS mock control group;

[0023] Figure 5 Figure B is the Lachnospira intervention PRV infection group;

[0024] Figure 5 Panel C in the figure is the PRV-infected group;

[0025] Figure 5 Figure D is a quantitative analysis.

[0026] Figure 6 : Effects of Lachnospira intervention on the transcriptional level of tight junctions in the lungs of PRV-infected mice.

[0027] Figure 6 Panel A shows the mRNA expression level of ZO-1 in lung tissues of PRV-infected mice induced by Lachnospira intervention;

[0028] Figure 6 Panel B shows the mRNA expression level of E-cadherin in lung tissues of PRV-infected mice induced by Lachnospira intervention;

[0029] Figure 6 Figure C shows the mRNA expression level of Occludin in lung tissues of PRV-infected mice treated with Lachnospira intervening;

[0030] Figure 6 Figure D shows the mRNA expression level of β-catenin in the lung tissue of PRV-infected mice intervened by Lachnospira.

[0031] Figure 7 : Effects of Lachnospira intervention on tight junction protein levels in the lungs of PRV-infected mice.

[0032] Figure 7 Figure A shows the expression level of ZO-1 protein in lung tissues of PRV-infected mice treated with Lachnospira;

[0033] Figure 7 Figure B shows the expression level of E-cadherin protein in the lung tissue of PRV-infected mice intervened by Lachnospira.

[0034] Figure 8 : Immunohistochemical detection of the effect of Lachnospira intervention on the expression levels of ZO-1 and E-cadherin in the lungs of PRV-infected mice.

[0035] Figure 8 Figures A, B, and C show the expression levels of ZO-1 in the lung tissues of mice in the PBS group, PRV group, and Lachnospira intervention PRV infection group, respectively;

[0036] Figure 8 Figures E, D, and F show the expression levels of E-cadherin in the lung tissues of mice in the PBS group, PRV group, and Lachnospira intervention PRV infection group, respectively.

[0037] Figure 9 : Gene set enrichment analysis (GSEA) plot.

[0038] Figure 9 Figure A shows the analysis of tight junction pathways in the lungs of mice treated with Lachnospira.

[0039] Figure 9 Panel B is an analysis of cell adhesion molecule pathways in the lungs of mice treated with Lachnospira;

[0040] Figure 9 Figure C is an analysis of the vascular smooth muscle contractile pathway in the lungs of mice treated with Lachnospira.

[0041] Figure 10 : Gene set enrichment analysis (GSEA) ridge plot.

[0042] Figure 11 :Verification of Lachnospira intervention on differentially expressed genes in the lung transcriptome of PRV-infected mice.

[0043] Figure 11 Figure A in the figure is the verification result of up-regulated differentially expressed genes;

[0044] Figure 11 Figure B is the verification result of down-regulated differentially expressed genes. DETAILED DESCRIPTION

[0045] First, mice were challenged with PRV. Feces were collected before challenge and on days 7 and 14 after challenge for 16S sequencing. Bioinformatics analysis identified bacteria with high abundance and significant changes in abundance as potential protectors against PRV infection. Ultimately, a bacterium (Lachnospiraceae) was identified that was upregulated on day 7 post-infection.

[0046] Example 1: Cultivation of strains

[0047] The original Lachnospira CLA-AA-M04 strain was purchased from Mingzhou Biological Co., Ltd., Ningbo, Zhejiang, China. It was cultured in BHI (tryptone 10 g / L, beef heart extract powder 17.5 g / L, sodium chloride 5.0 g / L, glucose 2.0 g / L, sodium hydrogen phosphate 2.5 g / L) medium (deoxygenated in advance) supplemented with 0.010 mL / mL Cystein-DTT (DL-Dithiothreitol)-solution (0.05 g / mL Cystein, 0.02 g / mL DTT) at 37°C under anaerobic conditions. When Lachnospira CLA-AA-M04 reached (OD 600 When the concentration of the bacterial strain (≈0.6) reached 108 CFU / mL, the mouse test was performed.

[0048] Example 2: Animal testing

[0049] Eight-week-old SPF female KM mice were randomly divided into three groups, with 16 mice in each group. They were divided into a control group, a pseudorabies virus (PRV) infection group, and a Lachnospira CLA-AA-M04 intervention group. Before PRV infection, the Lachnospira CLA-AA-M04 bacterial solution (10 6 CFU / g) for 4 days. The other groups of mice were treated with PBS buffer (100 μL). After 4 days, the mice were infected with PRV (157.7 TCID 50 During the experiment, mice continued to receive oral administration of the bacterial solution of the Lachnospira strain CLA-AA-M04, and mice in the PBS buffer group continued to receive oral administration of phosphate buffered saline (PBS).

[0050] Example 3: Detection of viral load

[0051] Four days after PRV infection, three surviving mice were randomly selected from each group and euthanized to collect lung and brain tissues. DNA was extracted from lung and brain tissues, and the PRV viral load in mouse tissues was detected by qPCR (conventional biological method) (PRVgB gene PRV-gH-F ACGTTCGGCTTCCTCTCC, PRV-gH-R GGTAGTCGTCGCTCTCGTG, PRV-gH-P FAMTCGCGCATCGTCTGGTGCAT-BHQ1). The results showed that the replication level of PRV in the lung and brain tissues of mice in the intervention group was significantly decreased (p<0.01) ( Figure 1 ), indicating that Lachnospira CLA-AA-M04 bacterial intervention can effectively inhibit the replication and spread of PRV.

[0052] Example 4: Statistics and analysis of mouse mortality

[0053] The survival period of infected mice was observed and a survival curve was drawn. The results showed that the survival rate of mice in the intervention group was higher than that in the control group (see Figure 2 ), further confirmed the positive role of Lachnospira CLA-AA-M04 in improving the host's ability to resist PRV infection.

[0054] Example 5: Pathological analysis of mouse lung tissue

[0055] Four days after PRV infection, three surviving mice were randomly selected from each group and euthanized. The lung tissues of the two groups of mice were collected for HE staining to analyze the pathological damage. The results showed that the lung structure of the mice in the PBS group was relatively intact, and no inflammatory cell infiltration was observed between the tissues. The lung tissues of the mice in the PRV infection group showed significant lesions, including pathological features such as alveolar wall thickening, inflammatory cell infiltration and local hemorrhage. In contrast, the degree of lung parenchymal damage in the mice in the Lachnospira intervention group was significantly reduced, and the number of inflammatory cells infiltrating in the interstitial tissues was significantly decreased (see Figure 3 ), indicating that the intervention of Lachnospira CLA-AA-M04 can effectively alleviate the lung inflammatory response caused by PRV infection.

[0056] Example 6: Detection of levels of key inflammatory factors TNF-α and IL-6 in mouse serum

[0057] Four days after PRV infection, three surviving mice were randomly selected from each group to remove their eyeballs and collect peripheral blood. Serum was then collected and ELISA kits were used to detect the levels of TNF-α (Accession No. GenBank: AK018578.1) and IL-6 (Accession No. GenBank: X51975.1). The results showed that the expression levels of TNF-α and IL-6 in the lung tissue and serum of mice in the Lachnospira intervention group were significantly lower than those in the PRV infection group ( Figure 4), suggesting that the intervention of Lachnospira CLA-AA-M04 can alleviate the systemic inflammatory response caused by PRV infection by inhibiting the overexpression of inflammatory factors.

[0058] Example 7: Analysis of barrier function of mice infected with PRV by Lachnospira intervening

[0059] Four days after PRV infection, three surviving mice were randomly selected from each group, and the integrity of the vascular barrier was assessed by tail vein injection of Evans blue dye. At the same time, three mice were randomly euthanized, and lung samples from the two groups of mice were collected. The expression of tight junction proteins (ZO-1 (accession number Protein_id: AAI38029.1), Occludin (accession number Protein_id: AAI38680.1) and adherens junction proteins (β-catenin (accession number Protein_id: AAA37280.1), E-cadherin (accession number Protein_id: AAH98501.1)) were analyzed by qPCR, Western Blot and immunohistochemistry. The experimental results showed that PRV infection significantly increased the penetration of Evans blue in lung tissue, indicating that the integrity of the respiratory epithelial barrier was damaged. However, the Evans blue penetration of mice in the Lachnospira CLA-AA-M04 intervention group was significantly lower than that in the PRV infection group ( Figure 5 ), indicating that Lachnospira CLA-AA-M04 can effectively restore the respiratory epithelial barrier damage caused by PRV infection.

[0060] Compared with the control group, the intervention of Lachnospira CLA-AA-M04 significantly upregulated the mRNA expression levels of ZO-1, Occludin, β-catenin and E-cadherin in the lung tissues of PRV-infected mice (see Figure 6 ) and the protein expression levels of ZO-1 and E-cadherin (see Figure 7 ).

[0061] The results of immunohistochemistry showed that more ZO-1 and E-cadherin proteins were identified in the lungs of mice treated with Lachnospira CLA-AA-M04 (see Figure 8 The above results indicate that Lachnospira CLA-AA-M04 intervention can effectively repair the respiratory epithelial barrier dysfunction caused by PRV infection.

[0062] Example 8: Analysis of transcriptome detection results of Lachnospira intervention in PRV-infected mouse lung tissue

[0063] Four days after PRV infection, three mice were randomly euthanized and lung samples from two groups of mice were collected for RNA-Seq analysis (Varet et al 2016). Using gene set enrichment analysis (GSEA) of differentially expressed genes (Subramanian et al 2005), it was found that tight junctions, cell adhesion molecules, and pathways related to vascular smooth muscle contraction were upregulated in the lungs of mice treated with Lachnospira CLA-AA-M04 compared with those after PRV infection ( Figure 9 GSEA analysis also showed that Lachnospira CLA-AA-M04 intervention significantly downregulated multiple pathways related to pathogen infection, including herpes virus infection-related pathways (see Figure 10 These results suggest that Lachnospira CLA-AA-M04 not only protects against respiratory tract infections by repairing the barrier function of the respiratory epithelium, but also may protect against viral replication and induced pathogenic infections by regulating the host immune response.

[0064] To verify the differential regulatory mechanism, DEGs of up-regulated / down-regulated pathways were randomly selected for qPCR validation. The results showed that the qPCR quantitative data were consistent with the transcriptome sequencing results (see Figure 11 ).

[0065] Main references:

[0066] 1. Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, Paulovich A, Pomeroy SL, Golub TR, Lander ES, Mesirov JP. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci USA, 2005.102(43):15545-15550.

[0067] 2. Varet H, Brillet-Guéguen L, Coppée JY, Dillies MA. SARTools: A DESeq2-and EdgeR-Based R Pipeline for Comprehensive Differential Analysis of RNA-SeqData. PLoS One, 2016.11(6):e0157022.

Claims

1. Use of Lachnospira CLA-AA-M04 in the preparation of a drug or preparation for preventing and treating pseudorabies virus infection.

2. The use according to claim 1, characterized in that The application is to reduce the virus load in the lung tissue and brain tissue of PRV-infected mice.

3. The use according to claim 1, characterized in that The application is to improve the survival rate of mice infected with PRV.

4. The use according to claim 1, characterized in that The Lachnospira CLA-AA-M04 can reduce pathological damage such as alveolar wall thickening and inflammatory infiltration in PRV-infected mice.

5. The use according to claim 1, characterized in that The application is to inhibit the expression of TNF-α and IL-6 in PRV-infected mice.

6. The use according to claim 1, characterized in that The application is to repair the permeability of the respiratory epithelial barrier of PRV-infected mice.

7. The use according to claim 1, characterized in that The application is to alleviate lung dysfunction caused by PRV infection by improving barrier function and regulating immune response.

Images