A Simple and Robust Approach for Evaluation of Antivirals Using a Recombinant Influenza Virus Expressing Gaussia Luciferase

Abstract

Influenza A virus (IAV) causes seasonal epidemics and occasional but devastating pandemics, which are major public health concerns. Because the effectiveness of seasonal vaccines is highly variable and the currently available drugs are limited in their efficacy because of the emergence of drug resistance, there is an urgent need to develop novel antivirals. In this study, we characterized a recombinant IAV-carrying Gaussia luciferase (Gluc) gene and determined its potential as a tool for evaluating therapeutics. We demonstrated that this recombinant IAV is replication-competent in tissue culture and pathogenic in mice, although it is slightly attenuated compared to the parental virus. Luciferase expression correlated well with virus propagation both in vitro and in vivo, providing a simple measure for viral replication in tissue culture and in mouse lungs. To demonstrate the utility of this virus, ribavirin and oseltamivir phosphate were used to treat the IAV-infected cells and mice, and we observed the dose-dependent inhibition of viral replication by a luciferase assay. Moreover, the decreased luciferase expression in the infected lungs could predict the protective efficacy of antiviral interventions as early as day 2 post virus challenge. In summary, this study provides a new and quantitative approach to evaluate antivirals against IAV.

Screening for Anti-Influenza Actives of Prefractionated Traditional Chinese Medicines

“…Previously, we generated a recombinant IAV expressing Gaussia luciferase, based on which a phenotypic high-throughput screening approach was subsequently established, providing a powerful tool for antiviral discovery [ 11 , 12 , 15 ]. A phenotypic screening was therefore carried out against the prefractionated TCM library for anti-influenza actives.…”

“…Madin–Darby canine kidney (MDCK) epithelial cells were grown in Dulbecco's modified Eagle's medium (DMEM; Cellgro, Manassas, VA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA), 1,000 units/mL penicillin and 100 μ g/mL of streptomycin (Invitrogen, Carlsbad, CA, USA). The replication-competent reporter influenza A virus carrying the Gaussia luciferase gene (PR8-PB2-Gluc) and wildtype influenza A/Puerto Rico/8/34 (H1N1, PR8) were propagated as previously described [ 11 , 12 , 15 ]. Infections were performed in Opti-MEM containing 1.5 µ g/mL N-tosyl-L-phenylalanine chloromethyl ketone (TPCK)-trypsin (Sigma-Aldrich, St. Louis, MO, USA).…”

“…In the present study, 100 medicinal plants which have been included in TCM prescriptions for antiviral treatment were selected and prefractionated into 5 fractions each. The library consisting of 500 prefractionated TCM extracts was subsequently subjected to a phenotypic screening for anti-influenza actives [ 11 , 12 ]. As a result, ten TCM fractions were identified to have antiviral potency against IAV, deserving further analysis for novel anti-influenza lead drugs.…”

A Mechanism Underlying Attenuation of Recombinant Influenza A Viruses Carrying Reporter Genes

“…Reporter virus PR8-NS1-Gluc and parental virus PR8 were generated and stocked as previously described [29]. Reporter virus PR8-PB2-Gluc was rescued with pDZ-PB2-Gluc along with the other seven IAV rescue plasmids pDZ-PA, -PB1, -NP, -HA, -NA, -M, and -NS1 as previously described [29]. The plasmid pDZ-PB2-Gluc, pPolI-NS and pPolI-NS-Gluc were kindly provided by Dr. Balaji Manicassamy (the University of Iowa).…”

“…Previously, we generated a replication-competent recombinant IAV carrying Gaussia luciferase (Gluc) PR8-NS1-Gluc, which shows a delay in replication kinetics and >1 log reduction of the virus titer [29]. In this study, we investigated the mechanism for attenuation of PR8-NS1-Gluc, and compared it to another reporter IAV PR8-Gluc (referred to as PR8-PB2-Gluc here) which was constructed using an updated strategy [24].…”