In Vitro and In Vivo Characterization of Novel Neuraminidase Substitutions in Influenza A(H1N1)pdm09 Virus Identified Using Laninamivir-Mediated In Vitro Selection

Abstract

Neuraminidase (NA) inhibitors (NAIs) are widely used antiviral drugs for the treatment of humans with influenza virus infections. There have been widespread reports of NAI resistance among seasonal A(H1N1) viruses, and most have been identified in oseltamivir-exposed patients or those treated with other NAIs. Thus, monitoring and identifying NA markers conferring resistance to NAIs-particularly newly introduced treatments-are critical to the management of viral infections. Therefore, we screened and identified substitutions conferring resistance to laninamivir by enriching random mutations in the NA gene of the 2009 pandemic influenza [A(H1N1)pdm09] virus followed by deep sequencing of the laninamivir-selected variants. After the generation of single mutants possessing each identified mutation, two A(H1N1)pdm09 recombinants possessing novel NA gene substitutions (i.e., D199E and P458T) were shown to exhibit resistance to more than one NAI. Of note, mutants possessing P458T-which is located outside of the catalytic or framework residue of the NA active site-exhibited highly reduced inhibition by all four approved NAIs. Using MDCK cells, we observed that the in vitro viral replication of the two recombinants was lower than that of the wild type (WT). Additionally, in infected mice, decreased mortality and/or mean lung viral titers were observed in mutants compared with the WT. Reverse mutations to the WT were observed in lung homogenate samples from D199E-infected mice after 3 serial passages. Overall, the novel NA substitutions identified could possibly emerge in influenza A(H1N1)pdm09 viruses during laninamivir therapy and the viruses could have altered NAI susceptibility, but the compromised in vitro/in vivo viral fitness may limit viral spreading.IMPORTANCE With the widespread emergence of NAI-resistant influenza virus strains, continuous monitoring of mutations that confer antiviral resistance is needed. Laninamivir is the most recently approved NAI in several countries; few data exist related to the in vitro selection of viral mutations conferring resistance to laninamivir. Thus, we screened and identified substitutions conferring resistance to laninamivir by random mutagenesis of the NA gene of the 2009 pandemic influenza [A(H1N1)pdm09] virus strain followed by deep sequencing of the laninamivir-selected variants. We found several novel substitutions in NA (D199E and P458T) in an A(H1N1)pdm09 background which conferred resistance to NAIs and which had an impact on viral fitness. Our study highlights the importance of continued surveillance for potential antiviral-resistant variants and the development of alternative therapeutics.

Keywords: influenza A virus; laninamivir; neuraminidase; pandemic H1N1 virus; resistance; viral fitness.

FIG 1

Schematic overview of in vitro and in vivo library screening methodology and characterization. A random mutant plasmid library was generated after introducing mutations in the six regions of the catalytic domain of A(H1N1)pdm09 NA. A reverse genetics technique was used to generate the random mutagenesis virus library, followed by passaging in MDCK cells with laninamivir selective pressure three times. The NA genes of the viruses that escaped the laninamivir selective pressure were analyzed by high-throughput sequencing. Selected mutations were introduced into the A(H1N1)pdm09 NA gene individually, and the impact of these substitutions on NAI susceptibility and viral fitness was evaluated.

FIG 2

NA protein expression and activity and enzyme kinetics of recombinant A(H1N1)pdm09 viruses. (A) The NA protein from recombinant virus-infected MDCK cells was evaluated using Western blot analysis. (B) NA activity was determined using a fluorogenic substrate (MUNANA)-based NA assay. (C) NA enzyme kinetic curves of the three mutant viruses at final MUNANA concentrations ranging from 0 to 1,000 μM. Fluorescence was quantified every 60 s for 60 min at 37°C with excitation and emission wavelengths of 360 and 460 nm, respectively. *, P < 0.05 compared with the value for the WT; ***, P < 0.001 compared with the value for the WT.

FIG 3

In vitro replication kinetics of recombinant A(H1N1)pdm09 viruses. MDCK cells were infected with mutant viruses at an MOI of 10⁻⁴. Cell culture supernatants were harvested at 12, 18, 24, 36, 48, and 60 h postinfection, and the titers were determined using TCID₅₀ assays in MDCK cells. *, P < 0.05 compared with the value for the WT; **, P < 0,001 compared with the value for the WT.

FIG 4

Pathogenicity of recombinant A(H1N1)pdm09 viruses in mice. Groups of BALB/c mice (n = 10/group) were infected with 10^4.0 TCID₅₀ (A, C, and E) and 10^3.0 TCID₅₀ (B, D, and F) of recombinant and wild-type virus. Mean body weight loss (A and B) and mortality (B and C) were monitored for 14 dpi. (C and D) Lung viral titers were determined at 2, 5, and 7 dpi using TCID₅₀ assays in MDCK cells. *, P < 0.01 compared with the value for the WT.