In addition, the HI assay does not distinguish between infectious and non-infectious disease particles. (PN) assay. Here we demonstrate that HA/NA pseudotypes mimic launch and access of influenza disease and that the PN assay exhibits good specificity and shows quantitative difference in neutralizing antibody titers against different H5N1 clades and subclades. Using immune ferret sera, we shown excellent correlation between the PN, MN, and HI assays. Therefore, we conclude the PN assay is definitely a sensitive and quantifiable method to measure neutralizing antibodies against varied clades and subclades of H5N1 influenza disease. Abbreviations: HA, hemagglutinin; NA, neuraminidase; HPAI, highly pathogenic avian influenza; MN, microneutralization; HI, hemagglutination inhibition; PN, pseudotype-based neutralization; RLA, relative luciferase activity Keywords: HPAI H5N1 disease, Pseudotype-based neutralization assay, Neutralizing antibodies 1.?Intro Since 1997, highly pathogenic avian influenza (HPAI) H5N1 viruses have been isolated from infected domestic poultry in numerous countries in Asia, Europe and Africa. As a result, an increasing quantity of avian to human being transmissions have occurred which is often with high mortality [1], [2]. Presently, HPAI H5N1 transmissions have been limited to avian to human being, however, continuous adaptation of the H5N1 disease or reassortment with seasonal human being influenza A strains may result in fresh H5N1 strains capable of efficient human-to-human transmission. As a result, these strains could cause an influenza pandemic with significant levels of morbidity and mortality. Neutralizing antibody reactions are critical for the prevention and clearance of influenza disease illness, and the measurement of neutralizing antibody reactions may be used for influenza serodiagnosis. Currently, the microneutralization (MN) and hemagglutination inhibition (HI) assays are used to estimate neutralizing antibody reactions against HPAI H5N1 viruses. The microneutralization (MN) assay confirmed by western blot analysis is considered to become the gold Zylofuramine standard for detecting anti-H5N1 specific neutralizing antibody response in humans [3], however, the assay is definitely somewhat labor-intensive and requires the use of numerous strains of replication qualified H5N1 viruses under biosafety level 3 (BSL-3) containment, which limits the common use of these assessments in many affected countries. Because it evaluates cytopathic effect (CPE) to reach an end-point titer, the MN assay, requires significant training to perform with consistent accuracy. The HI assay is based on the measurement of the ability of antibody to inhibit the hemmaglutination of erythocytes Zylofuramine by influenza viruses, and serves as a surrogate of neutralization of influenza computer virus. Unfortunately, the conventional HI assay that uses chicken erythrocytes as the indication cells utilized for seasonal influenza strains has been found to be poorly suited for use with avian H5N1 contamination [4]. Therefore, a altered HI assay using horse erythrocytes has been developed [5]. In addition, the HI assay does not distinguish between infectious and non-infectious computer virus particles. Therefore, the development of a standardized, quantifiable assay to measure anti-H5N1 neutralizing antibody responses, which does not require BSL-3 containment is usually urgently needed for in serodiagnosis of human HPAI infections as well as for vaccine evaluation. Lentiviral pseudotypes expressing heterologous glycoproteins from many viruses have been developed, including vesicular stomatitis computer virus (VSV), hepatitis C computer virus (HCV), the SARS coronavirus, Ebola, H7N1 avian influenza computer virus, H1N1 influenza computer virus, murine leukemia computer virus (MLV) and Lassa fever computer virus [6], [7], [8], [9], [10], [11], [12], [13]. These lentiviral pseudotypes have become useful tools in studies of viral access and release, anti-viral drug screening, serodiagnosis, and for use in assessing the response to vaccines. Recently, the development of lentiviral pseudotypes expressing H5HA or H5HA and N1NA has been reported [14], [15]. These H5HA or H5HA and N1NA pseudotypes undergo a single-round of contamination and cannot produce progeny viruses in the indication cell lines. Thus, assays based on pseudotyped particles do not require BSL-3 facilities for production Zylofuramine and screening. Pseudotype particles can also be designed to contain reporter genes, such as green fluorescent proteins (GFP) and luciferase genes. Therefore, infectivity (transduction efficiency) of these pseudotypes can be readily assessed by measuring fluorescence that is indicative of the expression of the reporter gene. In these studies, we generated a panel of pseudotype particles expressing HA and NA from H5N1 as well as H1N1 influenza viruses. Using the Fli1 panel we developed an influenza HA and NA pseudotype-based neutralization (PN) assay. We exhibited that HA and NA pseudotypes mimic wild type influenza A computer virus in their release and entry and that the PN assay is usually specific for clades and subclades of H5N1 influenza.