The Effect Of Influenza Viral Infection On Intestine And The Primary Study Of Its Cause On Diarrhea

Objectives It is found that there are some influenza virus infected patients have obvious symptom of diarrhea in clinical, but the exact mechanism is unknown. In this study, four different experimental animal models including influenza infection model, systemic inflammatory response model, viral antigen into the intestine model and intestinal flora model were established. To explore the directly cause of diarrhea induced by influenza virus, the changes of the microflora and inflammatory cells in the intestines of these mice in each animal model were detected. To explore the pathway that lead to diarrhea caused by the changes of intestinal flora and intestinal inflammatory after influenza virus infected, the expression of different inflammatory cells specific transcription factors in the peripheral immune organs and lungs were detected. This study is expected to help choose the optimum treatment method for flu patients, especially in children whose intestinal flora and immune System are immature.Materials and Methods 1. Influenza virus FM1 strain was amplified through chicken embryo allantoic cavity inoculation. The hemagglutination titers of influenza virus were measured and the virus stored at-80 ℃ for later use. 2. To establish influenza viral infected mouse model. Detect the LD50 and determine the optimal viral infection titers. 3. To establish systemic inflammatory response mouse model by tail vein injection with CD3 antibody and influenza virus antigen into the intestine model by intraperitoneal injection with viral antigens, observe the abnormal reaction of mice. 4. To establish intestinal microflora imbalance mouse model to determine appropriate types of antibiotics, dose and method of administration. 5. Compare the changes of physical signs of mice in each experimental group. 6. To culture and counts bacteria in mice feces and then analyze the changes in gut flora of mice in each group.7. To detect the expression of Th1, Th17 and Treg cell-specific transcription factors in peripheral immune organs, lung and intestinal tissue respectively using RT-PCR method. 8. To detect the expression and synthesis of IL-17 in intestinal tissue through RT-PCR and ELISA respectively.Results 1. The influenza virus FM1 strain was successfully amplified through chicken embryo allantoic cavity inoculation, and the hemagglutination titer of allantoic fluid was measured as 1:640. 2. Through the experiment, the LD50 of KM mice infected with influenza virus FM1 is 5×10-5.56, and 20% of the LD50 concentration was chosen as the dose for mice infection. 3. Via the tail vein injection with anti-mouse CD3 antibody(Systemic inflammatory response model), and intraperitoneal injection with influenza virus vaccine(viral antigens into the intestine model), all of the mice did not show significantly abnormal reaction. 4. The mice were given different dose of metronidazole, cephalosporin and neomycin through lavage separately, and then the changes of intestinal flora in each group of mice were compared. It was decided ultimately that mouse intestinal flora model would be established by 30 mg/ml of neomycin through lavage.. 5. After infected with influenza virus, the mice showed significantly decreased in body weight, hair looks dull, towering and curling hair, loss of appetite and other flu signs. The mice in systemic inflammation model group also showed weight loss, but there was no other significant change. 6. The bacteria in mice feces were cultured and counted, and it was found that in mice of virus infection model the Coli and enterococcus has significantly increased while the Lactobacillus and Bifidobacterium significantly reduced. The expression of Th17 cells specific transcription factor in the intestinal has significantly increased, indicating that the number of Th17 cells has significantly increased. However, the mice in normal control groups had no change. These results indicated that influenza infection cause changes in intestinal flora and intestinal inflammatory cells. 7. The peripheral immune organs and lungs of mice in viral infection and systemic inflammation groups were detected by RT-PCR. It’s shown that the expression of Th1 cellspecific transcription factor was significantly increased while the expression of Th17 cell-specific transcription factor was significantly decreased. The results proved that influenza viral infection induces a systemic inflammatory response in mice, and then lead to changes in the local intestinal inflammation and intestinal flora. 8. The results of detection about IL-17 expression level and IL-17 content in the intestinal tissue showed that both in viral control groups and systemic inflammatory response groups, the mice had been caused inflammatory response because of Th17 cells increased. This further illustrated that the changes in the intestinal of mice is due to systemic inflammatory response after infected with influenza.Conclusions Through analyzing the changes of intestinal flora and intestinal immune cells of mice in viral infection group, systemic inflammatory response group, viral antigens into the intestine and intestinal flora group model, it was illustrated that, the diarrhea induced by influenza infection was because of the changes in the intestinal flora and intestinal inflammatory cells. Furthermore, the changes of intestinal in mice caused by influenza virus were due to the migration of inflammatory cytokines and inflammatory cells, rather than the migration of viral antigens. This study also proved that CD3 antibody could be used to simulate systemic inflammatory response which is similar to that caused by influenza virus.