Journal of Clinical Virology 39 (2007) 27–33 Hemagglutinin pseudotyped lentiviral particles: Characterization of a new method for avian H5N1 influenza sero-diagnosis Isabelle Nefkens a,∗,1 , Jean-Michel Garcia a,1 , Chu Shui Ling a , Nad` ege Lagarde a , John Nicholls d , Dong Jiang Tang a , Malik Peiris b,∗∗ , Philippe Buchy c , Ralf Altmeyer a,2 a HKU-Pasteur Research Center, HongKong, SAR China b University of Hong Kong, Hong Kong, SAR China c Institut Pasteur in Cambodia, Phnom Penh, Cambodia d Departement of Pathology, University of Hong Kong, Hong Kong, SAR China Received 21 February 2007; accepted 21 February 2007 Abstract Background: Highly pathogenic avian influenza (HPAI) H5N1 has spread globally in birds and infected over 270 humans with an apparently high mortality rate. Serologic studies to determine the extent of asymptomatic H5N1 infection in humans and other mammals and to investigate the immunogenicity of current H5N1 vaccine candidates have been hampered by the biosafety requirements needed for H5N1 micro- neutralization tests. Objective: Development of a serodiagnostic tool for highly pathogenic influenza that reproduces H5N1 biology but can be used with less biohazard. Study Design: We have generated and evaluated H5 hemagglutinin pseudotyped lentiviral particles encoding the luciferase reporter (H5pp). Results: H5pp entry into target cells depends on 2-3 cell surface sialic acids and requires low pH for membrane fusion. H5pp infectivity is specifically neutralized by sera from patients and animals infected with H5N1 and correlates well with conventional microneutralization test. Conclusions: H5pp reproduce H5N1 influenza virus entry into target cells and potentially provides a high-throughput and safe method for sero-epidemiology. © 2007 Elsevier B.V. All rights reserved. Keywords: Influenza; Pseudotyped lentiviral particles; H5N1; Serodiagnostic Abbreviations: HI, hemagglutination inhibition; HA, hemagglutinin; H5, hemagglutinin of H5N1; H5pp, H5 pseudotyped viral particles; AMLV, amphotrophic envelope of the murine leukemia virus; sNA, soluble recombinant neuraminidase from Vibrio cholerae; SNA, Sambucus Nigra (elderberry) Bark lectin; MAA, Maackia amurensis lectin II; HPAI, highly pathogenic avian influenza; SA, sialic acids ∗ Corresponding author. Present address: CombinatoRx Singapore Pte Ltd., 11 Biopolis Way, Helios #08-05, Singapore 138667, Singapore.Tel.: +65 91515226. ∗∗ Corresponding author. E-mail addresses: isabelle.nefkens@gmail.com (I. Nefkens), malik@hkucc.hku.hk (M. Peiris). 1 Contributed equal to this work. 2 Present address: CombinatoRx Singapore Pte Ltd., 11 Biopolis Way, Helios #08-05, Singapore 138667, Singapore. 1. Introduction The global spread of highly pathogenic avian influenza A (HPAI) H5N1 viruses in poultry and its transmission to humans poses a pandemic threat. Since 2003 there have been 270 human cases with over 160 deaths (http://www. who.int/csr/disease/avian influenza/country). Binding of influenza virus to cellular receptors is determined by the viral hemagglutinin. The HA1 subunit of hemagglutinin binds to terminal sialic acids of glycoproteins and glycolipids at the cell surface (Skehel and Wiley, 2000; Skehel and Wiley, 2002). Avian influenza viruses preferentially bind 2-3-linked sialic acids (SA) (Skehel et al., 1982; Skehel et al., 1983, Russell et al., 2006) while human influenza viruses preferentially recognize 2-6-linked SA. Subsequent virus 1386-6532/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jcv.2007.02.005