Macropinocytosis:an Endocytic Pathway For H5n1 Subtype Avian Influenza Virus Infecting Cells In Vitro

Avian Influenza(AI) caused by Influenza A Virus was an important disease in poultry. So far, 16 types of hemagglutinin and 9 types of neuraminidase are identified in avian influenza virus. Based on their pathogenicity in chickens, avian influenza A viruses can be divided into viruses of high or low pathogenicity. All highly pathogenic avian influenza viruses known to date belong to the H5 or H7 subtype, however, only a small percentage of H5 and H7 viruses are low pathogenic. Since 1997, numerous outbreaks in poultry,which were accompanied by the occasional transmission of HPAI H5N1 viruses to humans, resulting in a case fatality rate of more than 50%, have been caused by highly pathogenic avian influenza viruses(HPAI) of the H5N1 subtype, which have led to the death or depopulation of significant numbers of chickens. Therefore, how to prevent HPAI H5N1 now becomes the major field of influenza study.Viruses must deliver their genome into the host cells to initiate replication. While some virus species can directly penetrate the plasma membrane and inject their genetic material into the cytoplasm(e.g. HIV), the majority of viruses enter cells via endocytosis. Due to the presence of multiple endocytic pathways in cells, determining the exact endocytic mechanisms used by viruses has been challenging. In order to discover pathways of H5N1 avian influenza virus entry, A/duck/Guangxi/35/2001(DK/35)(H5N1), rDK/35(Q226L/G228S)(H5N1), rDK35/HA-A160T(H5N1)was used as the main virus, and a noncompetitive inhibitor of dynamin’s GTPase activity dynasore were adopted to block the Dynamin-dependent, clathrin-mediated endocytosis and the Dynamin-dependent, caveolae-mediated endocytosis. A new dynasore-insensitive pathway became functional in the presence of serum. The specific inhibitor of macropinocytosis 5-(N-Ethyl-N-isopropyl) amiloride(EIPA) confirmed macropinocytosis may play an important role in the new pathway. Further, different kinds of cells and virus of different receptor binding affinity were detected. It indicates that the macropinocytosis entry exists in A549, MDCK, DF1 and different receptor binding affinity. The removal of the surface sialic acid receptors made the Dynamin-independent pathway ineffective, which means the pathway depend on sialic acid receptors.The macropinocytosis entry pathway can provide technique support for further study on thepathogenic and the control of influenza.