Study On The Inhibitory Mechanisms Of The Secondary Metabolite PQ Of Acid-resistant Fungus Against Influenza A Virus

Influenza virus is one of the early viruses that people study and it can spread rapidly and become pandemic in some regions or in the world in a short of time. Since 21st century, there are several influenza pandemics globally, such as the H5N1 avian influenza in 2005, the H1N1 Flu in 2009, and the recent H7N9 type A flu. The currently used antiviral drugs are M2 ion channel blockers and neuraminidase inhibitors(NAIs) which have been used worldwide. However, there have been resistant virus to these two kinds of anti-IAV drugs, so it is very important to develop novel anti-IAV drugs with new effect target.The receptor tyrosine kinase (RTKs) is a series of growth factors which are on the surface of the cells. They have significant influence on the growth, metabolism and differentiation of the cells. RTKs and their downstream signaling pathways have a close relationship with the infection and replication of influenza A virus(IAV). So RTKs and their downstream signaling pathways can become the new target spot for the discovery of the antiviral drugs.The secondary metabolites are the material which the microorganisms produce at the end of the logarithmic growth period and are unnecessary to the growth and reproduction of the microorganisms but they affect the differentiation of the microorganism and play an important role in the competition with other creatures. There are a lot of diversities of the secondary metabolites in chemical structure and biological activity, such as β-lactam and amino glycoside. It is very feasible to take advantage of the broad microorganism resources in China to discover new antiviral drugs with novel molecular structure and excellent biological activity. The research object of this thesis is a small bicyclic ketone compound PQ derived from the acid-resistant fungus Penicillium purpurogenum JS03-21 in Yunnan Province. Primary studies showed that PQ possessed good anti-IAV effects in vitro. This thesis aims to fully explore the anti-IAV mechanism of PQ in vitro and in vivo, in order to provide the theoretical reference for the development of the new IAV drugs.Herein, we firstly detected the anti-IAV effects of the secondary metabolite PQ of acid-resistant fungus by using the MDCK cell model and PR8 strain. We evaluated the antiviral effect of PQ by testing the cytopathic effect inhibition rate and the neuraminidase activity of the cell supernatant. The results indicated that the cytopathic effect and NA titer decreased significantly after PQ treatment, and the IC50 value was only 26.4μg/mL. Thus, PQ possessed good anti-IAV effects in vitro.We further explored the anti-IAV mechanisms of PQ in MDCK cells. We evaluated the expression and localization of virus nucleoproteins(NP) in MDCK cells at different time point by using immunofluorescence assay. The results showed that PQ treatment could delay the nucleus import and export process of virus NP protein, to decrease the production of influenza virus particles. The results of western blot indicated that PQ could inhibit the activation of key signal proteins in the RTK singling pathway which related to the replication of IAV, and consequently inhibit the the replication of IAV and virus induced inflammation response.We also investigated the inhibitory effects of PQ against the immune injury in IAV infected mice. Firstly, we established the mouse pneumonia model using Kunming mouse, and then detected the influence of PQ on the lung index, pulmonary virus titer, the content of cytokines in IAV infected mice and the death rate of mice. The results showed that PQ treatment could significantly decrease the secretion of inflammation factors, attenuate the pulmonary lesions, reduce the lung index and greatly improve the survival rate.In conclusion, this thesis deeply explored the anti-IAV mechanisms of the secondary metabolite PQ of acid-resistant fungus at the molecular, cellular and animal level using a variety of techniques and methods. The study revealed that PQ could inhibit IAV replication through the inhibition of activation of RTK pathway related to viral replication, suppression of viral replication and reduction of the secretion of cytokines related to inflammatory response. Therefore, this study indicated that the secondary metabolite PQ of acid-resistant fungus could inhibit IAV replication through targeting the RTK pathway, which was different from the previous ideas of developing anti-IAV drugs, and will provide the novel targets for drug screening and theoretical references for the development of novel anti-influenza virus drugs.