Profiling and characterization of influenza virus N1 strains potentially resistant to multiple neuraminidase inhibitors

Abstract

Neuraminidase inhibitors (NAIs) have been widely used to control influenza virus infection, but their increased use could promote the global emergence of resistant variants. Although various mutations associated with NAI resistance have been identified, the amino acid substitutions that confer multidrug resistance with undiminished viral fitness remain poorly understood. We therefore screened a known mutation(s) that could confer multidrug resistance to the currently approved NAIs oseltamivir, zanamivir, and peramivir by assessing recombinant viruses with mutant NA-encoding genes (catalytic residues R152K and R292K, framework residues E119A/D/G, D198N, H274Y, and N294S) in the backbones of the 2009 pandemic H1N1 (pH1N1) and highly pathogenic avian influenza (HPAI) H5N1 viruses. Of the 14 single and double mutant viruses recovered in the backbone of pH1N1, four variants (E119D, E119A/D/G-H274Y) exhibited reduced inhibition by all of the NAIs and two variants (E119D and E119D-H274Y) retained the overall properties of gene stability, replicative efficiency, pathogenicity, and transmissibility in vitro and in vivo. Of the nine recombinant H5N1 viruses, four variants (E119D, E119A/D/G-H274Y) also showed reduced inhibition by all of the NAIs, though their overall viral fitness was impaired in vitro and/or in vivo. Thus, single mutations or certain combination of the established mutations could confer potential multidrug resistance on pH1N1 or HPAI H5N1 viruses. Our findings emphasize the urgency of developing alternative drugs against influenza virus infection.

Importance: There has been a widespread emergence of influenza virus strains with reduced susceptibility to neuraminidase inhibitors (NAIs). We screened multidrug-resistant viruses by studying the viral fitness of neuraminidase mutants in vitro and in vivo. We found that recombinant E119D and E119A/D/G/-H274Y mutant viruses demonstrated reduced inhibition by all of the NAIs tested in both the backbone of the 2009 H1N1 pandemic (pH1N1) and highly pathogenic avian influenza H5N1 viruses. Furthermore, E119D and E119D-H274Y mutants in the pH1N1 background maintained overall fitness properties in vitro and in vivo. Our study highlights the importance of vigilance and continued surveillance of potential NAI multidrug-resistant influenza virus variants, as well as the development of alternative therapeutics.

FIG 1

Replication kinetics of recombinant pH1N1 viruses in vitro. Cells were infected with 12 recombinant viruses and WT pH1N1 at an MOI of 10⁻³. Cell culture supernatant were collected at 12, 24, 36, 48, 60, and 72 h postinfection, and titers were determined by measurement of log₁₀TCID₅₀/ml. (A and B) Growth curves of eight single mutants in MDCK cells. (C) Growth curves of four double mutants in MDCK cells. Error bars show the standard errors of the means obtained from three independent experiments. *, P < 0.05; **, P < 0.001 (compared with the value for WT pH1N1).

FIG 2

Replication kinetics of recombinant H5N1 viruses in vitro. Cells were infected with nine recombinant viruses and parental H5N1 at an MOI of 10⁻³. Cell culture supernatants were collected at 12, 24, 36, 48, 60, and 72 h postinfection, and titers were determined by measurement of log₁₀TCID₅₀/ml. (A and B) Growth curves of six single mutants in MDCK cells. (C) Growth curves of three double mutants in MDCK cells. Error bars show the standard errors of the means obtained from three independent experiments. *, P < 0.05 (compared with the value for WT pH1N1).

FIG 3

NA enzyme kinetics of the recombinant influenza viruses. Recombinant viruses in the pH1N1 (A and B) and H5N1 (C and D) backbones were standardized to equivalent doses, and the fluorogenic MUNANA substrate was used at final concentrations of 0 to 4,000 μM. Fluorescence was measured every 60 s for 1 h by using excitation and emission wavelengths of 355 and 460 nm, respectively.

FIG 4

Histopathologic lesions and immunohistochemistry in the lungs of infected ferrets. Ferrets intranasally infected with 10⁵ TCID₅₀ of pH1N1 virus and the E119D and E119D-H274Y mutants were euthanized at 5 dpi. (A) H&E staining of serial lung sections showing bronchointerstitial pneumonia. (B) Immunohistochemistry detecting viral NP in the lungs of ferrets infected with WT or E119D or E119D-H274Y mutant pH1N1 (original magnification, ×200).

FIG 5

Transmission of recombinant pH1N1 viruses via respiratory droplets in ferrets. Groups of four ferrets each were inoculated with 10^5.0 TCID₅₀ of WT or E119D or E119D-H274Y mutant pH1N1 virus intranasally (left side), and four naive ferrets were placed in the cages adjacent to the infected ferrets but separated by two stainless steel grids 3.5 cm apart at 1 dpi (right side). Nasal wash samples were collected from infected and contact ferrets, and titers were determined by measurement of EID₅₀/ml. Total numbers of positive exposed animals are shown. RD, respiratory droplet.

FIG 6

Daily monitoring of body temperatures of ferrets. Median changes in the body temperatures of ferrets infected with the WT pandemic (H1N1) 2009 virus (CA/04/09) or the E119D or E119D-H274Y mutant virus are shown. Naive contact animals were included and monitored daily for 12 days postinfection. Error bars indicate standard errors of the means. INF, infected; RD, respiratory droplet.

FIG 7

Molecular basis of the NA interaction with sialic acid and its inhibitors. The interactions of WT NA with sialic acid (A), oseltamivir (Oselt) (B), zanamivir (C), and peramivir (D) are illustrated by using the structures of Protein Data Bank (PDB) entries 4GZX, 2HU4, 3B7E, and 2HTU, respectively. The structure of the oseltamivir-resistant H274Y mutant and oseltamivir complex is presented by using PDB entry 3CL0. The structures of E119D and H274Y double mutation complexes with zanamivir and peramivir were generated from the WT structures by using the Coot program (49). PDB entry 3CL0 was used to generate the double mutation structure with oseltamivir by using the same program. The residues critical for the interaction between NA and ligands are shown.