动物造模,甲型流感病毒 z-bio 2023-03-04 904

　　Objective: To compare the two subtypes of influenza A virus H1N1 strains A/Shanghai/37T/2009(H1N1)(pdm09) clinical isolates and A/PuertoRico/8/34(H1N1)(PR8) standard strains infected with C57BL/6 small Changes in the pathological state and the induction of immune responses of specific CD8+ T cells.

　　Methods: ① Wild-type C57BL/6 mice were infected with PR8 and pdm09 viruses at 9.85×106 half tissue culture infective dose (50% tissue culture infective dose, TCID50), respectively, and their status was observed and recorded every day (for 14 consecutive days). Mortality. Mice were sacrificed on the 1st and 3rd day after infection, and their lungs were weighed to calculate lung index; the left lobe lung was fixed with 4% paraformaldehyde and then stained with H&E (hematoxylin-eosin staining) to observe the lungs after infection. Pathological changes; real-time quantitative PCR (qRT-PCR) was used to detect the pulmonary viral load. ② Mice were infected with PR8 and pdm09 with 0.25 times the median lethal dose (50%, LD50), respectively, and the mice were weighed daily for continuous observation. Lungs and spleens were dissected on the 8th day after infection, and the pathological changes of the lungs were compared by H&E staining; flow cytometry (FACS) was used to detect the proportion of virus-specific CD8+ T cells in the spleen, as well as IFN-γ, TNF-α, The expression of cytokines such as IL-2, granzyme B and immunosuppressive checkpoint molecules PD-1, LAG-3, Tim-3 and CTLA-4.

　　Results: ①After being infected with the same TCID50 influenza virus, both groups of mice developed disease state. All mice in the PR8 group died on the 5th day of infection, and the mortality rate was significantly higher than that in the pdm09 group (P<0.01). The results of index and viral load in the PR8 group were significantly higher than those in the pdm09 group; H&E staining showed that the PR8 group had severe pathological damage, thickened alveolar walls, lung parenchyma, and increased blood cell exudation, while the pdm09 group had pathological damage to the lung tissue of the mice. Mild and less inflammatory infiltration; ②After infection with the same LD50 influenza virus, the weight loss and lung tissue pathological damage of the mice in the PR8 group were more serious than those in the pdm09 group; flow cytometry results showed that the virus-specific CD8+ T cells induced in the PR8 group The ratio of IFN-γ, IL-2 and TNF-α expressed by activated CD8+ T cells (CD8+CD44High) in the PR8 group was significantly lower than that in the pdm09 group; It expressed more immunosuppressive molecules PD-1 and LAG-3.

　　Conclusion: Compared with pdm09, PR8 can cause more serious damage to mice, which may be due to the functional exhaustion of its activated specific CD8+ T cells, resulting in impaired specific immune protection and decreased ability to resist and clear viruses. cause more serious pathological damage.