title: In vitro evaluation of neuraminidase inhibitors using the neuraminidase-dependent release assay of hemagglutinin-pseudotyped viruses.
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 pseudotype 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 fluorogenic substrate 2'-(4-methylumbelliferyl)-alpha-d-N-acetylneuraminic acid (MUNANA) and also correlated with the infectivity results.
authors.affiliation: Genomics Research Center, Academia Sinica, 128 Academia Road, Section 2, Taipei 115, Taiwan.
authors.forename: Ching-Yao
authors.initials: CY
authors.lastname: Su
authors.forename: Shi-Yun
authors.initials: SY
authors.lastname: Wang
authors.forename: Jiun-Jie
authors.initials: JJ
authors.lastname: Shie
authors.forename: King-Song
authors.initials: KS
authors.lastname: Jeng
authors.forename: Nigel J
authors.initials: NJ
authors.lastname: Temperton
authors.forename: Jim-Min
authors.initials: JM
authors.lastname: Fang
authors.forename: Chi-Huey
authors.initials: CH
authors.lastname: Wong
authors.forename: Yih-Shyun E
authors.initials: YS
authors.lastname: Cheng
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D002460:Cell Line; D002642:Chick Embryo; D004353:Drug Evaluation, Preclinical; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D017930:Genes, Reporter / Q000187:drug effects; D005818:Genetic Engineering; D006389:Hemagglutinins, Viral / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007251:Influenza, Human / Q000188:drug therapy*; D009052:Leukemia Virus, Murine / Q000235:genetics / Q000378:metabolism; D009439:Neuraminidase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D009975:Orthomyxoviridae / Q000187:drug effects* / Q000201:enzymology / Q000502:physiology; D053139:Oseltamivir / Q000494:pharmacology; D017201:Virus Shedding / Q000187:drug effects; D053243:Zanamivir / Q000494:pharmacology
chemical_list: D000998:Antiviral Agents; D004791:Enzyme Inhibitors; D006389:Hemagglutinins, Viral; D053139:Oseltamivir; D009439:Neuraminidase; D053243:Zanamivir