Detection of molecular markers of drug resistance in 2009 pandemic influenza A (H1N1) viruses by pyrosequencing

Abstract

The M2 blockers amantadine and rimantadine and the neuraminidase (NA) inhibitors (NAIs) oseltamivir and zanamivir are approved by the FDA for use for the control of influenza A virus infections. The 2009 pandemic influenza A (H1N1) viruses (H1N1pdm) are reassortants that acquired M and NA gene segments from a Eurasian adamantane-resistant swine influenza virus. NAI resistance in the H1N1pdm viruses has been rare, and its occurrence is mainly limited to oseltamivir-exposed patients. The pyrosequencing assay has been proven to be a useful tool in surveillance for drug resistance in seasonal influenza A viruses. We provide a protocol which allows the detection of adamantane resistance markers as well as the I43T change, which is unique to the H1N1pdm M2 protein. The protocol also allows the detection of changes at residues V116, I117, E119, Q136, K150, D151, D199, I223, H275, and N295 in the NA, known to alter NAI drug susceptibility. We report on the detection of the first cases of the oseltamivir resistance-conferring mutation H275Y and the I223V change in viruses from the United States using the approach described in this study. Moreover, the assay permits the quick identification of the major NA group (V106/N248, I106/D248, or I106/N248) to which a pandemic virus belongs. Pyrosequencing is well suited for the detection of drug resistance markers and signature mutations in the M and NA gene segments of the pandemic H1N1 influenza viruses.

FIG. 1.

The variability in M2 protein-coding sequences among influenza A viruses can be used to differentiate between the various subtypes. (A) Partial nucleotide sequence alignment of the M2 protein-coding region from viruses of different subtypes: a pandemic virus, A/California/04/2009; a triple-reassortant swine influenza virus, A/Ohio/01/2007; seasonal H1N1 and H3N2 viruses, A/Minnesota/04/2009 and A/Pennsylvania/05/2009, respectively; and an H5N1 virus, A/Vietnam/1203/2004. (B) Partial amino acid sequence alignment of the M2 protein. Highlighted are amino acids 26 through 43. (C to H) Analysis of the sequences at residue 43. Seasonal H1N1 virus shows the presence of ATT (isoleucine) at residue 43 (I43) (C). Seasonal H3N2 virus, avian H5N1 virus, and a triple-reassortant swine H1N2 virus contain CTT (leucine) at this position, which corresponds to residue L43 (D, E, and H). Another triple-reassortant swine H1N1 virus has TTT (phenylalanine) at this position (G). The pandemic viruses have the unique sequence ACT (threonine) at residue 43 (F).

FIG. 2.

Detection of molecular markers for oseltamivir resistance among the H1N1pdm viruses. (A) The wild-type CAC (histidine) sequence was detected in A/Texas/41/2009 virus. The oseltamivir resistance-conferring mutation CAC (histidine) to TAC (tyrosine) was detected in A/Washington/29/2009 (B) and A/North Carolina/16/2009 (C). A mixture of wild-type sequence CAC (histidine) and oseltamivir resistance-conferring mutation TAC (tyrosine) was detected in A/Utah/34/2009 (D). (E to G) Detection of changes at I223 (I222). Wild-type sequences ATA (isoleucine) at codon 223 (I222) were present, as expected, in both A/Tennessee/08/2009 and the triple-reassortant swine influenza A/Ohio/01/2007 viruses. However, the change from ATA (isoleucine) to GTA (valine) at residue 223 (residue 222) was detected in A/North Carolina/15/2009 virus (G).

FIG. 3.

Analysis of sequences at residues 106 and 247 for distinction between the NAs from the three major groups of the pandemic H1N1 viruses. (A) Valine (GTA) at position 106 in A/Texas/41/2009 virus; (B) mutation from GTA to ATA (isoleucine) at the same residue in viral isolate A/North Dakota/04/2009; (C) A/Texas/41/2009 with AAT (asparagine) at position 248 (N247); (D) change from AAT to GAT (aspartic acid) at the same residue, N248D (N247D), in A/North Dakota/04/2009 virus.