Adaption Of Human Influenza H3N2Virus In A Mouse Pneumonitis Model And Inhibition Of Viral Replication By Yinqiao Powder

Objective:1. Observe the adaptation process of Influenza A H3N2influenza virus (A/Aichi/2/68, H3N2) on BALB/c mouse lung model.2. Establish a control method of Yinqiao powder freeze-dried sample, provide the material basis to explore the mechanism of inhibition of influenza virus by Yinqiao powder.3. Inspect the antiviral effect of Yinqiao powder in vitro.4. Explore the inhibition of viral replication by Yinqiao powder in vivo.Methods:1. The human influenza virus strains A/Aichi/2/68A (H3N2) as a research object, compared with survival status of mouse, pulmonary index, the virus titer and the hemagglutination titer of each generation, to find the changes of adaption strains between the wild strain and the mutation strain.2. High performance liquid chromatography (HPLC) was used to determinate the quality of the honeysuckle, Arctium, glycyrrhizic acid.3. Determined the antiviral effect by cell culture. Evaluated potency inhibition by the cytopathic effect (CPE) and the plaque reduction test method under different experimental strategies (treatment, protection)4. We use BALB/c mice which were intranasal inoculated with H3N2strain of influenza A virus, to observe the living conditions, determinate lung index, to observed pathological changes by hematoxylin-eosin staining (HE staining), the alveolar ultrastructure changes by transmission electron microscopy; the mouse lung suspension were detected the quantity of infectious virus titer by reduced plaque test and the nucleoprotein (NP) expression by Western blot.Results:1. In the passage process of the mouse lung, the HA of human influenza virus H3N2changes to adapt to the new mouse host, with the125amino acid point of NS1protein mutations, the strain have more replication capacity, along with the viral titer increased, the mice began to appear towering hair, curled up, diet reduced, lack of exercise, shortness of breath, eating and drinking plummeted, weight loss and other symptoms of infection, mouse lungs emerge more and more severe edema, congestion and lung hepatization.2. The quality control system of Yinqiao powder freeze-dried samples has good stability and repeatability, the chlorogenic acid, arctiin and licorice ammonium of this sample met the required of the national pharmacopoeia.3. In vitro inhibition experiments, various concentrations of Yinqiao powder samples had better suppress the virus and cell survival effect (P<0.01) in the mode of treatment and protection; plaque reduction test results show that SI of two modes were39.31and43.48respectively, can effectively inhibit influenza virus, in line with the results of the CPE.4. In vivo experimental results show that the Yinqiaosan sample can reduce the systemic symptoms, lung index, the scope of the rat lung lesions, alveolar space exudate, alveolar ultrastructure change and virus particle number; reduce mouse lung virus titers and mouse lung expression of nucleoprotein (nucleoprotein, NP).Conclusion:1. The adapted strains model provides an research tool to understanding the adaption of type A influenza virus cross-species, not only described the probably rocess of wild-type influenza virus strains in the new host adaptation, lay groundwork to explore the virus and its host the molecular changes in the process of adaptation, but also establish a platform for subsequent pharmacological studies, it has great significance for the evaluation antiviral effect and mechanism of drug treatment on H3N2influenza virus.2. Yinqiao powder freeze-dried samples can effectively protect the active ingredient in the decoction, and compared to conventional formulations, get more easier standard quantitative, handling more suitable for the pharmacology and mechanism studies.3. Yinqiao powder samples has anti-influenza A H3N2virus effect in vitro.4. Yinqiao powder sample has inhibit replication of A type influenza virus H3N2in vivo, the major role is at the process of viral replication.