Predicting Permissive Mutations That Improve the Fitness of A(H1N1)pdm09 Viruses Bearing the H275Y Neuraminidase Substitution

Abstract

Oseltamivir-resistant influenza viruses arise due to amino acid mutations in key residues of the viral neuraminidase (NA). These changes often come at a fitness cost; however, it is known that permissive mutations in the viral NA can overcome this cost. This result was observed in former seasonal A(H1N1) viruses in 2007 which expressed the H275Y substitution (N1 numbering) with no apparent fitness cost and lead to widespread oseltamivir resistance. Therefore, this study aims to predict permissive mutations that may similarly enable fit H275Y variants to arise in currently circulating A(H1N1)pdm09 viruses. The first approach in this study utilized in silico analyses to predict potentially permissive mutations. The second approach involved the generation of a virus library which encompassed all possible NA mutations while keeping H275Y fixed. Fit variants were then selected by serially passaging the virus library either through ferrets by transmission or passaging once in vitro. The fitness impact of selected substitutions was further evaluated experimentally. The computational approach predicted three candidate permissive NA mutations which, in combination with each other, restored the replicative fitness of an H275Y variant. The second approach identified a stringent bottleneck during transmission between ferrets; however, three further substitutions were identified which may improve transmissibility. A comparison of fit H275Y variants in vitro and in experimentally infected animals showed a statistically significant correlation in the variants that were positively selected. Overall, this study provides valuable tools and insights into potential permissive mutations that may facilitate the emergence of a fit H275Y A(H1N1)pdm09 variant. IMPORTANCE Oseltamivir (Tamiflu) is the most widely used antiviral for the treatment of influenza infections. Therefore, resistance to oseltamivir is a public health concern. This study is important as it explores the different evolutionary pathways available to current circulating influenza viruses that may lead to widespread oseltamivir resistance. Specifically, this study develops valuable experimental and computational tools to evaluate the fitness landscape of circulating A(H1N1)pmd09 influenza viruses bearing the H275Y mutation. The H275Y substitution is most commonly reported to confer oseltamivir resistance but also leads to loss of virus replication and transmission fitness, which limits its spread. However, it is known from previous influenza seasons that influenza viruses can evolve to overcome this loss of fitness. Therefore, this study aims to prospectively predict how contemporary A(H1N1)pmd09 influenza viruses may evolve to overcome the fitness cost of bearing the H275Y NA substitution, which could result in widespread oseltamivir resistance.

Keywords: antivirals; influenza; oseltamivir; resistance.

FIG 1

Testing the impact of candidate substitutions derived from computational approaches on NA enzyme function and virus replication. (A) The NA glycoprotein of A/South Australia/16/2017 was mutated such that it expressed the H275Y substitution by itself or in different combinations with candidate permissive substitutions. The proteins were expressed following transfection of 293T cells, and the relative NA activity and expression was calculated as a percentage of the wild-type NA protein. Experiments were performed in duplicate on two separate occasions, and data are expressed as the mean ± SD. The relative NA activity and expression for NA proteins containing candidate substitutions were compared against that of the H275Y-NA using a Student’s unpaired two-tailed t test. *, P < 0.05; **, P < 0.01; WT, wild type. (B) The replication kinetics of reverse genetic viruses, namely, SA16-H275Y, SA16-WT, and SA16-H275Y+S95N+S286G+S299A, was assessed in A549 cells following infection at an MOI of 0.1. The experiment was performed in triplicates and viral titers at each time point measured using a Student’s unpaired two-tailed t test. *, P < 0.05; **, P < 0.01.

FIG 2

Schematic of the transmission model used to select for fit H275Y variants in the ferret model of influenza infection. Codon-based mutagenesis and reverse genetics were used to generate virus libraries, such that they contained viruses with all possible codon mutations in the A/South Australia/16/2017-NA while H275Y remained fixed. Virus libraries generated on three independent occasions were pooled to increase the likelihood that all codon mutations were represented. The combined library was passaged through ferrets via serial transmission (n = 4 independent lines of transmission), and nasal wash samples were collected and analyzed to determine if any variant was selected via passage through ferrets. As a control, the A/South Australia/16/2017-H275Y virus (control) was also generated by reverse genetics and passaged once through ferrets to determine the background mutation frequency.

FIG 3

Deep sequencing of plasmid and virus libraries (and relevant controls), as well as ferret nasal wash samples, and viral supernatants after in vitro infection was done to determine the per-codon mutation frequency, composition, and fraction of total mutations sampled in the viral NA. (A) Libraries comprised multinucleotide (2 or 3) codon mutations, whereas the controls did not. Compared with the experimental infection of ferrets, most viruses in direct contact 2 and aerosol contact animals contained single-nucleotide codon mutations. (B) Viruses from ferret nasal wash samples generally contained a greater ratio of synonymous changes to nonsynonymous changes, indicating purifying selection. (C) The fraction of multinucleotide mutations that were observed multiple times in the samples, after combining biological replicates, was >90% in the plasmid and virus libraries and was substantially reduced in ferret nasal wash samples and in viral supernatants after in vitro infection.

FIG 4

Viral titers and variant frequencies in nasal wash samples from ferrets experimentally infected with the NA-H275Y virus library and from ferrets subsequently infected via transmission. (A) At 24 h postinoculation, experimentally infected animals were cohoused with direct contact 1 ferret. Nasal wash samples from direct contact 1 ferrets were monitored for infection, and on the day that influenza infection was confirmed, they were cohoused with direct contact 2 ferrets. Nasal wash samples from direct contact 2 ferrets were monitored for infection, and on the day that influenza infection was confirmed, they were placed in a cage adjacent to aerosol contacts. All animals were nasal washed daily during the experiment, and infectious virus was detected in nasal wash samples from animals along the transmission chain. For each animal, a single time point (black arrows) was selected for analysis by deep sequencing. (B) NGS data were aligned using Bowtie2, and variants observed at a greater than 1% frequency were called using VarScan, where the average read depth at each site was >10,000 and P values for variant calls above 1% were <0.05 for all called positions. A different set of variants were detected in each transmission chain, and most variants were not detected beyond direct contact 1 animals. However, substitutions I188T, K386Q, and S388L were also detected in direct contact 2 animals.

FIG 5

Impact of candidate substitutions identified following passage of virus libraries in ferrets on NA enzyme function and virus replication. (A) The NA glycoprotein of the A/South Australia/16/2017 virus was mutated such that it contained the H275Y substitution by itself or in combination with candidate permissive substitutions. The proteins were expressed following transfection of 293T cells, and the relative NA activity and expression were calculated as a percentage of wild-type (WT) NA protein (lacking any substitution). The assay was performed in duplicate on three independent occasions, and the mean ± SD are shown. The relative NA activity and expression for the NA proteins containing candidate substitutions was compared against that of the H275Y-NA using a Student’s unpaired two-tailed t test. *, P < 0.05; **, P < 0.01. (B) The replication kinetics of reverse genetics viruses, namely, SA16-H275Y, SA16-WT, and SA16-H275Y modified with either I188T, K386Q, or S388L NA substitution, was assessed in A549 cells following infection at an MOI of 0.1. The experiment was performed in triplicates, and viral titers at each time point were measured using a Student’s unpaired two-tailed t test. *, P < 0.05; **, P < 0.01.

FIG 6

The mutational tolerance of the H275Y-NA glycoprotein in different settings. (A) The amino acid preference (enrichment of amino acid) at each site was calculated from the three replicates of in vitro passaging and was used to calculate Shannon entropy (mutational tolerance), which is visualized on the NA monomer using PyMOL (PDB 4B7R). (B) The amino acid preference was calculated from the four replicated of experimentally infected animals, and the Shannon entropy at each site is visualized on the NA monomer. (C) NA sequences from the GISAID acid frequency at each site was used to calculate mutational tolerance at each site. (D) A scatterplot showing correlation between amino acid preferences after in vitro replication and amino acid preferences after in vivo replication (Pearson’s r = 0.51, P < 0.01).

FIG 7

The positions of previously identified permissive substitutions and candidate permissive substitutions identified in our studies have been visualized on a N1 NA monomer from an A(H1N1)pdm09 virus (PDB 4B7R) in complex with oseltamivir. The variability of amino acids at sites of interest was calculated after alignment of all full-length N1 protein sequences from the GISAID database (24,463 sequences). Amino acids are represented on a color scale of blue (low variability) to red (high variability). Oseltamivir is represented as a ligand (cyan), and the two calcium ions in the crystal structure are represented by yellow dots. The positions of previously identified substitutions in N1 NAs, namely, V241I and N369K, are highlighted in red.