Antiviral Research 79 (2008) 199–205 Contents lists available at ScienceDirect Antiviral Research journal homepage: www.elsevier.com/locate/antiviral In vitro evaluation of neuraminidase inhibitors using the neuraminidase-dependent release assay of hemagglutinin-pseudotyped viruses Ching-Yao Su a,b , Shi-Yun Wang a , Jiun-Jie Shie a , King-Song Jeng c , Nigel J. Temperton d , Jim-Min Fang a,e , Chi-Huey Wong a,b , Yih-Shyun E. Cheng a,∗ a Genomics Research Center, Academia Sinica, 128 Academia Road, Section 2, Taipei 115, Taiwan, ROC b Institute of Biochemical Sciences, National Taiwan University, 1 Roosevelt Road, Section 4, Taipei 106, Taiwan, ROC c Institute of Molecular Biology, Academia Sinica, 128 Academia Road, Section 2, Taipei 115, Taiwan, ROC d MRC/UCL Centre for Medical Molecular Virology, Division of Infection and Immunity, University College London, 46 Cleveland Street, London W1T4JF, United Kingdom e Department of Chemistry, National Taiwan University, 1 Roosevelt Road, Section 4, Taipei 106, Taiwan, ROC article info Article history: Received 29 October 2007 Accepted 17 March 2008 Keywords: Neuraminidase inhibitor Pseudotype virus Antiviral Viral release assay Drug resistance abstract For the treatment of influenza virus infections, neuraminidase inhibitors (NAIs) that prevent the release of virus particles have been effective against most influenza strains. Several neuraminidase (NA) assays are available for the evaluation of NAIs. To understand the NAI functions under physiological conditions, assays mimicking viral particle release should be useful. We have constructed retrovirus-based reporter viruses that are pseudotyped with hemagglutinin (HA) glycoprotein by transfection of producer cells using plasmids expressing retroviral gag-pol, influenza HA, NA, and firefly luciferase genes. Similarly to the life cycle of influenza viruses, the release of pseudotype viruses also requires neuraminidase functions. This requirement was used to develop an assay to evaluate NAI activities by measuring inhibition of pseudo- type virus production at different NAI concentrations. The pseudotype virus release assay was used to determine the IC 50 values of Oseltamivir carboxylate, Zanamivir, and the novel phosphonate congeners of Oseltamivir against N1 group neuraminidases and their H274Y Oseltamivir carboxylate-resistant mutants. The deduced IC 50 values obtained using the release assay correlated with those determined using the fluo- rogenic substrate 2 ′ -(4-methylumbelliferyl)--d-N-acetylneuraminic acid (MUNANA) and also correlated with the infectivity results. © 2008 Elsevier B.V. All rights reserved. 1. Introduction Influenza virus infects avian and many species of mammals. Seasonal human infections by influenza viruses result in mortality and financial losses and are managed predominantly by vaccina- tion (Chowell et al., 2007; Vila-C ´ orcoles et al., 2007). The recent H5N1 avian influenza viruses that are highly pathogenic to several wild and domestic avian species infected more than 300 persons, many fatally, in many Asian countries and raised the concern of another influenza pandemic (Hampson, 2006; Poland et al., 2007). Among the therapeutic options for the treatment and preven- tion of influenza infections, neuraminidase inhibitors (NAI) are most promising (Alymova et al., 2005; De Clercq, 2006). Before an effective vaccine for avian influenza is developed, NAIs are crucial ∗ Corresponding author. Tel.: +886 2 2789 9930x334; fax: +886 2 2789 9931. E-mail address: ysecheng@gate.sinica.edu.tw (Y.-S.E. Cheng). ammunition for protection against the possible pandemic H5N1 viruses that are often resistant to the other class of approved anti- flu agents: adamantanamine derivatives (Gani et al., 2005; Hurt et al., 2007). Several neuraminidase assays are currently used for the evalu- ation of NAIs. The substrates of these assays are usually synthetic sialic acid derivatives that upon cleavage by neuraminidase produce convenient reporting signals for the measurements (Buxton et al., 2000; Cabezas et al., 1983; Franc ¸ a de Barros et al., 2003; Onodera, 1994). In order to understand NAI inhibition under physiologi- cal conditions, cell-based assays are helpful as the neuraminidase reaction does not simply involve the terminal sialic acid of the sub- strates. The physiological functions of influenza neuraminidase are thought to enhance the spread of the influenza infection in sev- eral ways. After replication, packaging, and budding of progeny viruses, the neuraminidases are required to cleave the cell surface sialic acids to release the progeny virus from infected cells (Palese et al., 1974). The neuraminidase also prevents the aggregation of 0166-3542/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.antiviral.2008.03.002