Objective To construct pseudotype retrovirus which integrates hemagglutinin(HA)of H5N1 avian influenza virus(AIV)isolated from human in Shenzhen.Methods AIV HA gene was amplified bv RT-PCR,then it was ligated with pGEM-T vector,and identified by restriction enzyme digestion and sequenced.HA gene was cloned into CMV/R vector at the site of Sal Ⅰ and BamH Ⅰ.pHR-Luc,pCMV&8.2 and CMV-HA were co-transfected into 293T cell by co-precipitation with calcium phosphate.The pseudotype virus supernatant was harvested 72 h post-transfection and ultracentrifugation,and the HA and P24 expression on the surface of pseudotype virus was analyzed by western blot.Meanwhile.the infection activity of HIV-HA pseudotype virus was identified in different kinds of cell lines,including MDCK,HeLa,CHO and 293T.Results A/Shenzhen/406H/06 belonged to subclade2.3 with open reading frame(ORF)of HA gene encoded 567 amino acides,whose accession number was EF137706 in GenBank.HA gene was cloned into CMV/R successfully.After co-transfection of above vectors,it revealed that HA protein could integrate pseudotype virus by western blot,and precursor protein HA0 could cleavage into HA1 and HA2 with biological activity.Finally.HIV-HA pseudotype virus could infect 4 kinds of cell lines,which indicated its property of infectivity and catholicity.Conclusion The pseudotype retrnvirns wassuccessfully constructed,which can integrate HA protein of A/Shenzhen/406H/06 and had property of infectivity.It call be used in the further research,including selection of neutralizing antibodies and epitope analysis.

Objective To establish neutralization assay based on H5N1 avian influenza pseudotyped virus in vitro and to evaluate neutralizing titer of convalescent serum from 2 patients with H5N1 avian influenza.Methods pHR-Luc,pCMV△8.2 and CMV/R-SH or CMV/R-TH were cotransfected into 293T cell by co-precipitation with calcium phosphate.Pseudotyped virus supernatant was harvested 72 h posttranofection and identified the expression of HA and P24 by Western blot,and then we analyzed infective activity of 200 μL supernatant of pseudotyped virus.293T cell integrated HA was prepared and anti-HA antibodies in convalescent serum were measured with FACS assay.Neutralizing titers of convalescent serums against Shenzhen and Thailand pseudotyped virus were determined based on calculating IC50 with neutralizing assay.Results Pseudotyped virus involved P24 and HA,and precursor protein HA0 could cleavage into HA1 and HA2 with biological activity.Pseudotyped virus possessed better infective activity,and RLA value was about 2 × 104 with 200 μL supernatant.Both convalescent serums contained anti-HA antibodies and had cross-reactivity against different virus clades with FACS assay.Both convalescent serums had neutralizingactivity and could cross-neutralize different virus clades.However,both serums'neutralizing titers against Shenzhen virus were higher than Thailand.Conclusion We successfully constructed infectious pseudotyped virus which integrated HA of Shenzhen or Thailand virus,and it could be used for evaluation of serum neutralizing activity fast,efficiently and safely with broadly application prospect.

To establish a method based on a pseudotyped virus assay for screening drugs for anti-H5N1 avian influenza virus, and then to evaluate anti-virus activity of traditional Chinese herbal compounds with the function of purgation, detoxification, cooling the blood and reinforcing the healthy qi based on seropharmacology.