Construction And Characterization Of Pseudovirus Bearing The Hemagglutinin Of Avian Influenza H5N1 Virus

Since 2003, the human disease due to highly pathogenic avian influenza H5N1 virus has elicited global concern. However, the experiment with handling live avian influenza virus, particularly the neutralization assay, is restricted in Biosafety Level 3 laboratory facility or higher which is not always available.The envelope glycoprotein HA on influenza virus is the most important antigen that initiates neutralizing antibody response in host and also involved in viral replication. Via HA, influenza viruses bind to cellular receptor. The pseudovirus bearing HA can bind to target cells while losing the ability of replication. Therefore, it can be a surrogate virus for the serological diagnosis of avian influenza virus and other relevant studies.The main purpose of our study is to construct pseudovirus bearing H5N1 HA based on a lentivirus vector system. Then we study the biological feature of the pseudovirus. With the newly established viral particles, we performed the serological tests. Our findings are the following:1. Three eukaryotic expression plasmids, pLP-HA-AH1, pLP-HA-VN1194 and pLP-HA-AHOpti were successfully constructed. The expression of HA protein in 293T cells was detected by IFA and immunohistochemistry staining.2. Pseudovirus bearing the HA of human H5N1 viruses was generated by the co-transfection of either of the above plasmids and the packaging plasmids pLP1, pLp2, pEmGFP. The morphology of the pseudoviruses was observed by the electronic microscope. HA protein on the pseudovirus was detected by Western Blot.3. Characterization of the pseudoviruses3.1 All of the pseudovirus Vp-HA-AH1, Vp-HA-AHOpti, Vp-VN1194 and Vp-VSVG can infect 293T, MDCK and BEAS-2B cells,and all the pseudoviruses can agglutinate the red blood cells.3.2. The HA gene from different sources affect the efficiency of the packaging of the pseudovirus.The packaging efficiency of the pseudovirus from HA gene of A/Vietnam/1194/2004 (H5N1) is higher than the packaging efficiency from A/Anhui/1/2005 (H5Nl).But the optimized HA gene can not obviously improve packaging efficiency of the pseudovirus.3.3 Our neutralization assay by using the pseudoviruses was comparable with the conventional microneutralization (MN) assay with wild-type viruses. A high degree of correlation, sensitivity and specificity was detected.In conclusion, pseudoviruses coated with HA of highly pathogenic avian influenzaH5N1 viruses were successfully constructed. In addition, the assay is safe and can beperformed in the high-throughput diagnosis of highly pathogenic avian influenza virus,evaluation of vaccine and mechanism study.