Emergence of Oseltamivir-Resistant Pandemic (H1N1) 2009 Virus within 48 Hours

An oseltamivir-resistant influenza A pandemic (H1N1) 2009 virus evolved and emerged from zero to 52% of detectable virus within 48 hours of a patient’s exposure to oseltamivir. Phylogenetic analysis and data gathered by pyrosequencing and cloning directly on clinical samples suggest that the mutant emerged de novo.


RT-PCR was performed with the SuperScript III
reverse transcription at 55°C for 10 min and initial denaturation at 94°C for 2.5 min; followed by 40 cycles of denaturation at 94°C for 32 s, annealing at 57°C for 76 s, and extension at 68°C for 33 s; and a final extension at 68°C for 5 min. PCR products were analyzed by gel electrophoresis to estimate the yield.

Pyrosequencing
Pyrosequencing was performed according to the manufacturer's guidelines (Biotage, Uppsala, Sweden). Briefly, ≈200 ng of biotinylated PCR product was reacted with streptavidincoated beads (GE Healthcare, Little Chalfont, UK) by shaking at room temperature for 15 min, followed by collecting DNA-coated beads by vacuum onto a 96-well vacuum tool and serially immersing the beads into 70% ethanol, 0·2 mol/L NaOH, and 10 mmol/L Tris-acetate, pH 7.6, washing buffer. The beads with single-stranded DNA template were released into each well of PSQ96-well plate (Biotage) with 40 µL of annealing buffer (20 mmol/L Tris-acetate, pH 7·6, and 2 mmol/L magnesium acetate) containing the sequencing primer (5'-TAGAATCAGGATAACAGGAGCA-3') at a final concentration of 0.4 μmol/L. The plate was heated at 80ºC for 2 min and then cooled to room temperature for 10 min before being placed into the PyroMark Q96 ID System (Biotage). The sequencing procedure was performed at room temperature by cyclic dispensation of substrates, enzymes, and 4 dNTPs (Biotage) in a prespecified order to enable single nucleotide polymorphism analysis and generation of quantitative data. We sequenced a 25-bp region that included the H275Y mutation of NA.
Relative proportions of bases, expressed as a percentage, were determined by using the PyroMark instrument.
The description of the mutation as H275Y means that the mutation in the gene results in the original amino acid at position 275 along NA (in N1 numbering) changing from the expected wild-type histidine (H) to tyrosine (Y). The actual change in the NA gene is a single point mutation from a cytosine base to a thymidine base.

Phylogenetic Analysis
All 7 strains with the NA H275Y mutation for which the whole genome (10 genes from 8 segments) was available from the National Center for Biotechnology Information (Bethesda, MD, USA) influenza virus resource (2) were compared. A drug-sensitive isolate (ON141), which was geographically and temporally closely related (a difference of <1 week) with full genome available, was also included. Because the nucleotide level also enables seeing synonymous nucleotide exchanges (without an amino acid change) that could harbor additional similarity information, we chose to derive a maximum likelihood tree over the whole coding genome to investigate the relationship of these 10 strains in further detail. Nucleotide alignments of the coding regions of all 10 genes in the 8 segments of the 10 strains under study were concatenated.  Technical Appendix Figure. Maximum-likelihood phylogenetic tree. Shaded strains are oseltamivir susceptible (Singapore/ON129) and resistant (Singapore/GN285) isolates from the case presented.
Singapore/ON141 is a drug-sensitive isolate geographically and temporally closely related to ON129 and GN285 is shown in italics. The remaining strains in the tree are all isolates with the H275Y mutation.
Scale bar indicates nucleotide substitutions per site.