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.

Keywords: Gaussia luciferase; antiviral; influenza A virus; therapeutics.

Figure 1

In vitro characterization of PR8–NS1–Gluc. (a–c) Madin-Darby canine kidney (MDCK) cells were infected with PR8–NS1–Gluc and influenza A virus (IAV)–PR8 at a multiplicity of infection (MOI) of 0.01 and incubated with N-tosyl-L-phenylalanine chloromethyl ketone (TPCK)–trypsin for the indicated times. Aliquotes were removed for determination of (a) viral titers and (b) Gaussia luciferase activity. (c) The correlation between supernatant luminescence and infectious virus titers of IAV were fit by linear regression using GraphPad Prism 5 (La Jolla, CA, USA) (R² = 0.807, p < 0.0001). (d) Gaussia luciferase signals derived from supernatants of virus-infected cells at MOI of 0.001, 0.01, or 0.1. * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 2

PR8–NS1–Gluc is virulent in mice. (a,b) BALB/c mice were intranasally inoculated with indicated doses of PR8–NS1–Gluc virus (n = 10 in each group). The body weight (a) and survival (b) were monitored daily. (c,d) BALB/c mice (n = 3) were infected with 10³ TCID₅₀ of PR8–NS1–Gluc or were mock infected. Three days after infection, the indicated organs were collected, and the levels of luciferase in these organs were determined (c). Six days after infection, the lung tissue sections were collected for hematoxylin and eosin staining (d). *** p < 0.0001.

Figure 3

Kinetics of PR8–NS1–Gluc spread and clearance in lungs of BALB/c mice. Mice were intranasally infected with 10³ or 10⁵ TCID₅₀ of PR8–NS1–Gluc. At the indicated times, lungs were collected from infected animals, and the amounts of luciferase and viral titers were determined. The correlation between the two variants were fit by linear regression using GraphPad Prism 5. (a–c) Data correspond to the time course for the dose of 10³ TCID₅₀. (d–f) Data correspond to the time course for the dose of 10⁵ TCID₅₀. The R² and p-values for the linear regression analysis are indicated on each graph.

Figure 4

In vitro antiviral determination using PR8–NS1–Gluc as a tool. Madin-Darby canine kidney (MDCK) cells were infected with PR8–NS1–Gluc at a multiplicity of infection (MOI) of 0.01 and incubated with N-tosyl-L-phenylalanine chloromethyl ketone (TPCK)–trypsin as well as increasing concentrations of ribavirin (a) or oseltamivir phosphate (b) for 24 h. Supernatants were collected for determination of Gaussia luciferase activity, and the inhibitory effects were analyzed using GraphPad Prism 5.

Figure 5

In vivo evaluation of therapeutic interventions using PR8–NS1–Gluc as a tool. Mice were intranasally infected with 10³ TCID₅₀ of PR8–NS1–Gluc and were treated with indicated drugs by gavage (n = 8 in each group). The treatments were started 2 h before infection and were given twice daily until mice were sacrificed. At days 2 and 4 after infection, four mice in each group were dissected and the Gaussia luciferase level (a) and viral load (b), respectively, in infected lungs were determined (ns: no significance; * p < 0.05; ** p < 0.01; *** p < 0.001).