Study On The Blocking Effect Of Lactobacillus Plantarum 0111 Strain On H9N2 Subtype Avian Influenza Virus

Probiotics are widely used to promote and improve the health of humans and animals,and can function through a variety of mechanisms.They can prevent the colonization of pathogens,block when pathogens invade cells or adhere to their surfaces,and have a regulatory effect on the host's immune system.It has been proven to prevent and reduce rotavirus infection,alleviate antibiotic-related diarrhea,and alleviate diseases caused by lactose intolerance.Lactic acid bacteria have been shown to have antiviral effects,such as respiratory syncytial virus and transmissible gastroenteritis virus.The colonization ability of Lactobacillus plantarum in the intestine makes it play the role of maintaining the balance of intestinal flora and promoting the intestinal immune response.It is a microorganism with probiotic effect in the intestine.Therefore,the identification of new types of probiotics with antiviral effects can help prevent the invasion of adaptive viruses.The H9N2 subtype AIV can not only infect poultry,but also humans,and has caused human deaths.The first H9N2 subtype AIV was isolated in turkeys in the 1960s,and it became widespread subsequently.Nowadays,H9N2 subtype AIV is still the main type of avian influenza,which seriously affects the breeding industry in my country.This study explored the protective effect of Lactobacillus plantarum 0111 against H9N2subtype AIV.The specific research content and results are as follows:(1)Establish a mouse infection model and randomly divide the mice into 3 groups:experimental group(0111+H9N2),infection control group(PBS+H9N2)and normal control group(PBS+PBS).After continuously feeding Lactobacillus plantarum 0111for one week,mouse serum and alveolar lavage fluid were collected for multi-factor determination of type I interferon;lungs and intestines were collected for fluorescence quantitative PCR to analyze the transcription level of type I interferon and ISGs;Separate single cells from lung and alveolar lavage fluid,and use flow cytometry to analyze NK cells.The mouse spleen,mesenteric lymph nodes and Peyer's collective lymph nodes were collected on the 10th day of feeding.Flow cytometric analysis of CD3~+CD4~+and CD3~+CD8~+T cell content changes in the spleen and mesenteric lymph nodes;CD3~+CD4~+IFN-?~+cells,CD3~+CD4~+IL-4~+cells and CD3~+CD4~+TNF~+cells Changes;CD3~+CD8~+IFN-?~+cells and CD3~+CD8~+TNF~+cells content changes.At the same time,detect the changes in the content of B220~+Ig A~+cells in Peyer's collection lymph nodes,and the changes in the expression of dendritic fine surface molecules CD80 and CD86.The H9N2 subtype AIV was subsequently challenged.After the challenge,the weight change and survival status of the mice were monitored daily.The lungs and jejunum of the mice were collected and fixed one week after the challenge for pathological analysis.Stool collected and stored at-80°C.Collect lung and alveolar lavage fluid,use multi-factor detection to analyze inflammatory cytokines IL-6 and TNF-?,and store part of lungs at-80°C for virus titer determination.14 days after the challenge,Pyle's collection of lymph nodes was collected,and the content of B220+Ig A+cells was determined by flow cytometry.The results show that Lactobacillus plantarum 0111 has a good effect on improving the immunity of mice,and regulates the innate immune response and adaptive immune response.Reduce tissue damage caused by H9N2 subtype AIV and reduce virus titer.At the same time,Lactobacillus plantarum significantly reduces the expression of inflammatory cytokines TNF-?and IL-6.(2)Use the A549 cell platform to establish an in vitro cell infection model.There are three groups:the experimental group(0111+H9N2),the infection control group(DMEM+H9N2)and the normal control group(Normal).After 2 hours of co-cultivation of Lactobacillus plantarum and A549 cells,fluorescence real-time quantitative PCR was used to detect the transcription levels of type I interferons and ISGs.After the H9N2 subtype AIV is challenged,the cell status is checked daily,and the virus growth curve is drawn.After the CCK8 method is used,the virus inhibition rate is calculated.The results show that pretreatment of A549 cells by Lactobacillus plantarum can significantly reduce the virus titer and increase the transcription levels of IFN-?,ISG15 and MX1,which are consistent with the results of animal model experiments.(3)The feces collected in the mouse animal model are subjected to DNA extraction and high-throughput sequencing.The results showed that the invasion of H9N2 subtype AIV made the intestinal flora imbalance and the intestinal microbial structure shifted.Feeding Lactobacillus plantarum 0111 can effectively improve the imbalance of intestinal flora caused by H9N2 subtype AIV,enhance the stability of intestinal flora,and enhance the important role of intestinal symbiotic microorganisms in maintaining intestinal homeostasis,which is important for the future Laid the foundation for the development of anti-influenza microecological preparations and anti-influenza drugs In this study,Lactobacillus plantarum 0111 was used to explore the effect of probiotics against H9N2 subtype AIV.The effects of Lactobacillus plantarum on the innate immunity,adaptive immunity and intestinal microecosystem of mice were verified through in vivo and in vitro experiments.This research lays the foundation for the development of anti-influenza microecological preparations and anti-influenza drugs in the future.