Preliminary Study On The Correlation And Mechanism Between Intestinal Microbiota Disorder And BVDV Infection Based On A Mouse Model

Bovine viral diarrhea-mucosal disease(BVD-MD)is an important infectious disease of cattle caused by bovine viral diarrhea virus(BVDV)infection.It is one of the key quarantine diseases in large-scale cattle farms and international trade,causing significant damage to the cattle industry.Currently,there is no effective drug to prevent or treat BVD-MD,and vaccination is the main strategy for disease control.However,due to the variability of the virus strains and the diversity of BVDV types,the efficacy of vaccine is not satisfactory.Therefore,in-depth study of the pathogenic molecular mechanism of BVDV is still a major research direction,which is of great significance for the prevention and control of this disease.Gut microbiota is a diverse and abundant microbial community in the body,which participates in host digestion,nutrient absorption,and immune regulation.In recent years,growing evidence has shown that gut microbiota is closely related to the occurrence of various diseases,and the development of microbiota-targeted drugs is highly favored.Despite the growing understanding of the association between gut microbiota and various diseases,its relevance to BVDV infection has not been reported.In this study,the authors used a BVDV-infected mouse model to investigate the impact of virus infection on gut microbiota composition.Fecal samples from mice infected with BVDV for 7 days were collected,and 16S rRNA sequencing was performed.The results showed that BVDV infection could lead to a decrease in gut microbiota alpha diversity,but there was no significant statistical difference.The beta diversity bar chart analysis showed that the gut microbiota composition in the BVDV infection group was different from the control group,with a decrease in Firmicutes and an increase in Bacteroidetes.Analysis of the main microbial taxa at the family and genus levels found that BVDV infection could lead to a decrease in the relative abundance of short-chain fatty acid-producing bacteria.Further analysis of species with a relative abundance of less than 1%found that BVDV infection could significantly affect the composition and relative abundance of low-abundance microbiota.The results suggest that BVDV infection can change gut microbiota composition,but the correlation may not be significant due to the susceptibility of the animal model.To further investigate the causal relationship between gut microbiota and BVDV infection,the authors developed an antibiotic-induced gut microbiota dysbiosis mouse model to explore the effect of gut microbiota dysbiosis on BVDV susceptibility.After 21 days of antibiotic treatment,Fecal samples from mice treated with antibiotics for 0 and 9 days were collected and subjected to 16 S rRNA sequencing.The results showed that antibiotic treatment significantly reduced gut microbiota alpha diversity,and the Venn diagram also confirmed the effect of antibiotics on gut microbiota diversity.Analysis of specific species found that the relative abundance of Firmicutes and Bacteroidetes in the antibiotic group was significantly reduced,while the relative abundance of Proteobacteria was significantly increased.Furthermore,the antibiotic treatment significantly reduced the abundance of aerobic and anaerobic bacteria and Gram-positive bacteria,while increasing the abundance of Gram-negative bacteria and potential pathogenic bacteria.Based on previous reports and our experimental results,we successfully constructed an antibiotic-induced gut microbiota dysbiosis mouse model.In addition,analysis of the gut microbiota at 9 days after the termination of antibiotic treatment found that the gut microbiota composition had significantly recovered compared to day 0,but it was still different from that of healthy controls,indicating that gut microbiota remained dysbiotic during BVDV infection.Based on the established gut microbiota dysbiosis mouse model,the authors further investigated the impact of dysbiosis on BVDV susceptibility.At day 7 post-BVDV infection,the authors collected the duodenum,jejunum,liver,and spleen from mice and used q RT-PCR and Western blot to detect the expression level of BVDV RNA and protein.The results showed that gut microbiota dysbiosis significantly increased the levels of BVDV RNA and protein in the duodenum,jejunum,spleen,and liver of mice.To further clarify the role of gut microbiota on BVDV susceptibility and the feasibility of intervening in viral infection by modulation of gut microbiota,the authors performed fecal microbiota transplantation(FMT)experiments and found that FMT treatment significantly inhibited the virus load in the duodenum,jejunum,spleen,and liver of mice compared to the antibiotic-BVDV group.These results indicate that gut microbiota dysbiosis can significantly increase BVDV susceptibility.To further elucidate the potential molecular mechanism by which gut microbiota dysbiosis increases BVDV susceptibility,the authors tested the innate immune-related regulatory molecules and T lymphocyte proliferation and apoptosis level mediated by BVDV.The results showed that gut microbiota dysbiosis significantly reduced the levels of IFN-αand IFN-βcompared to the BVDV group,while FMT treatment significantly increased the levels of IFN-αand IFN-β.Further analysis of the upstream regulatory molecules of IFN-I showed that gut microbiota could significantly regulate the expression of IRF1,IRF3,IRF7,TLR7,and TLR9,but had no significant effect on TLR3 and RIG-I levels.Given that previous studies have shown that the proliferation and apoptosis level of peripheral blood lymphocytes(PBL)in mice are involved in the pathogenesis of BVDV,the authors collected peripheral blood lymphocytes from BVDV-infected mice at day 7 and found that gut microbiota dysbiosis significantly reduced the number of CD3~+,CD4~+,and CD8~+T lymphocytes and inhibiting PBL proliferation,however,while FMT significantly restored the number of CD3~+and CD8~+T lymphocytes,The proliferation level of PBL has also been significantly improved.To further explore the potential mechanism,the authors tested the levels of PBL proliferation and apoptosis and found that gut microbiota could significantly affect PBL proliferation and apoptosis via regulation of the PI3K/Akt,caspase 9/caspase 3,and ERK pathways.In summary,this study focused on the potential impact of BVDV infection on the gut microbiota using a mouse model.By developing a gut microbiota dysbiosis mouse model and combining FMT experiments,the authors further clarified the role of gut microbiota dysbiosis in increasing BVDV susceptibility.The main detection targets were duodenum and PBL,revealing the molecular mechanisms underlying IFN-I and its upstream regulatory molecules,as well as PBL proliferation,apoptosis,and increased susceptibility to BVDV due to gut microbiota disorders.The study elucidated novel insights into the molecular mechanisms of BVDV pathogenesis and suggested that gut microbiota is a potential target for the development of antiviral therapies.