Purification Of H5N1 Hemagglutinin Expressed By Recombinant Vaccinia Viruses And Kinetics Of Anti-Oseltamivir Neuraminidase

Highly pathogenic avian influenza virus H5N1 could break the host range restriction for the transmission from avian to human.Human-to-human transmission remains extremely rare and it has not yet circulated in the human population.The existence of host restriction mainly relies on the characteristics of hemagglutinin receptor binding.Recent gene analysis indicated that some H5N1 strains isolated from infected patients got changes at the hemagglutinin receptor binding site.The first part of the research aims to expose the realities that the H5 HA has been undergoing the"human adaptation"changing receptor binding characteristics to obtain the potential for transmission among human population.Firstly we introduced the mutations A134V,I151F,E186D and I151F+A134V+E186D by site-directed mutagenesis,which forming or near the receptor binding site.Then we rescued recombinant influenza viruses containing those mutations by reverse genetics system.Two mutations A134V and E186D could keep stable after the passages in egg allantoic lumans.We got the crystals from the purified HAs protein.And the crystals of HA-LSTa/LSTc complexes got disfracted,which supported the hypothesis that E186D could enable the avian H5 HA binding to human receptors.On the other hand,A134V did not have this ability,remaining preferencially binding avian receptors. Interestingly,the I151F mutation converted to F151L under the circumstances of intoduced into the eggs,which strongly suggested the possibilities that this mutation might abolish the binding of avian receptors.Controversely,I151F might be related with the shift of receptor binding specificity from avian to human.As a result,we adopted the generation of recombinant vaccinia viruses to express the HA I151F and H5 I151F+A134V+E186D.For the first time the crystals generated from highly purified H5 I151F membrane protein was available for further analysing the HA-receptor interactions from the structural perspective and glycan microarray technology,which pave the way for investigation of the possibilities of human to human transmission caused by avian influenza viruses.To prepare for controlling next influenza pandemic,many countries have compiled huge NAI(neuraminidase inhibitors:oseltamivir)stock.Because of the wild use of the drug,the resistance of Oseltamivir has become more and more challenging.Previous studies have been focused on the mutations H274Y,H253Y,N294S of N1.In this study we pay more attention to the amino acid substitutions I117V and I314V.Those two mutations were firstly found in H5N1 viruses isolated from sick chickens,which could reduce the sensitivity of Oseltamivir.The questions arised that if the human infected this kind of viruses, could the doctors still use the Oseltamivir for antiviral treatment? Then we generated the mutated A/Vietnam/1203/04(H5N1)viruses with N1 Il17V,I314V,I117V+I314V respectively on the WSN background by the method of reverse genetics.According to the measurement of NA activities,it was found that the Ki of N1 I117V and N1 I117V+I314V to Oseltamivir increased slightly, which means they can reduce the sensitivity of Oseltamivir,which might be related with I117V residue indirect influencing binding of the drug by sitting beside E119,one of the consensus of NA catalytic site.