Sars Coronavirus, The H5n1 Avian Influenza Virus Pathogenesis Of Research

Mammalian expression of codon optimized proteinsThe mammalian expression system has been widely used in recent years due to its ability of producing correctly folded and glycosylated protein.However,one disadvange of this system is the low protein yield.This paper described a new method of codon optimization,which enhances protein expression to a higher level compared with non-optimized sequence.The establishment of a stable expression cell line enables the production of protein in large scale.In order to simplify protein purification,the expression vector was modified by inserting a CD5 lead sequence upstream of coding sequence,which will enhance protein secretion into supernatant,and a human Fc tag,which will facilitate protein purification with a Protein A column.In addition,in order to obtain proteins without tags,a TEV site was constructed between coding sequence and Fc tag.This system enables production of lage-scale,high-purity,and glycosylated proteins.Mechanism of SARS-CoV entry host cellsThe entry pathway of SARS-CoV into host cells has been controversial,direct membrane was firstly observed by electro-micorscope in 2003,however,later,it was reported that the entry is dependent on pH,which suggests that an endocytic pathway may also exist.Our paper found that SARS-CoV can enter via endocytosis and the SARS-CoV was targeted to endosome in Vero E6 cells.We also found that the entry pathway of SARS-CoV into host cells did not dependent on clathrin or caveolae,but, lipid rafts,an important microdomains rich in cholesterol was found to be involved in this process.These findings are of great importance to the development of novel anti-viral drugs.Molecular mechanism of H5N1 avian influenza induced acute lung injuryRecent outbreaks of highly pathogenic avian influenza H5N1 in poultry and humans have raised the concern that a pandemic will occur in the near future.According to the most recent World Health Organization data,reported on March 11,2009,the cumulative mortality attributed to avian influenza HSN1 virus infection is greater than 60%.Acute lung injury(ALI) is the major manifestation associated with patients hospitalized with H5N1 influenza infection.ALI has a high mortality rate and,other than mechanical ventilation and supportive clinical care,there are few specific therapeutic options of proven benefit.We know little about the molecular pathogenesis of ALI.This paper found that H5N1 can cause ALI via two pathways:1.H5N1 induce lung macrophages to produce ROS,which will oxidize phospholipids and generate OxPLs,which will then activate TLR4-TRIF-NFkB pathway,producing large quantity of cytokines,inducing ALI.ROS inhibitors,antibodies that can bind OxPLs can alievate the severity of ALI.2.H5N1 viruses induce autophagy in lung epithelial cells via Akt-TSC2-mTOR pathway.This autophagy leads to cell death,which will cause lung injury.Specific inhibitors and siRNAs targeting autophagy will alievate the severity of ALI.These findings expand our understanding of H5N1 induced lung injury and also provide valuable information for the anti-viral research.Mechanism of H5N1 avian influenza entry host cellsThe mechanism of influenza virus entry has been widely studied;however,all the studies have been carried out with virus strain H1N1,and most of the studies have used the canine kidney cell line MDCK.It has been reported that the influenza virus can enter cells via clathrin-mediated endocytosis,caveolae-mediated endocytosis,or non-clathrin-, non-caveolae-dependent endocytosis,depending on the different cell lines or virus strains used.However,since the risk of an H5N1 pandemic is increasing due to the widespread occurrence of birds infected with H5N1 and the lack of effective drugs or vaccines,it is imperative to understand the mechanism of disease caused by H5N1 in humans.In this paper,we used the H5N1 strain and a cell line of human lung tissue origin,which is the main target of H5N1 infection,and found that the H5N1 virus can enter a human lung carcinoma cell line via clathrin-dependent endocytosis.This finding is relevant for the development of novel therapeutic drugs targeting influenza virus H5N1.