Detection of Molecular Markers of Drug Resistance in 2009 Pandemic Influenza A (H1N1) Viruses by Pyrosequencing

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.In the spring of 2009, an antigenically novel influenza A virus (H1N1) was detected in North America (7). The rapid widespread transmission of the virus resulted in the declaration of an influenza pandemic by the World Health Organization (WHO) (42). The 2009 pandemic influenza A (H1N1) virus (H1N1pdm) was determined to be a reassortant with a combination of gene segments that had not been previously described (12, 21). Phylogenetic analysis of the full genome sequences revealed that in the late 1990s, reassortment between seasonal influenza A virus (H3N2), classical swine influenza virus, and North American avian influenza viruses led to the appearance of triple-reassortant H3N2 and H1N2 swine influenza viruses that have since circulated in pigs in North America (40). The pandemic virus was a result of further reassortment between a triple-reassortant swine influenza virus and a Eurasian avian influenza virus-like swine influenza virus, resulting in the acquisition of two gene segments, coding for the M protein and neuraminidase (NA), from the Eurasian avian influenza virus-like swine influenza virus lineage. Recent genome sequence analysis performed with pandemic viruses collected in different regions found variants with characteristic amino acid changes, including 2 amino acid changes in the NA (21, 29). The reports identified three NA variants among the H1N1pdm viruses: one variant group has V106 and N248 (referred to as the A/California/04/2009 group); the second variant, named the A/Osaka/164/2009 group, is characterized by I106 and N248; and the third NA variant group contains I106 and D248, such as the A/New York/18/2009 strain.Currently circulating triple-reassortant swine influenza viruses in the United States do not contain any known markers of adamantane resistance (L26F, V27A, A30V, A30T, S31N, and G34E) (10, 25), whereas the Eurasian avian-like influenza viruses as well as the pandemic virus contain the adamantane resistance-conferring change S31N in the M2 protein. Currently, two classes of antiviral drugs are approved for use by the FDA for the control of influenza virus infections: adamantanes (M2 blockers) and neuraminidase inhibitors (NAIs). Resistance to adamantanes makes the NAIs oseltamivir and zanamivir the only pharmaceutical options available for use for the control of infections caused by the pandemic virus. Monitoring of resistance to NAIs is mainly based on the NA inhibition assay (23, 39, 41), which allows the detection of resistance conferred by known and novel mutations. However, the NA inhibition assay requires virus isolation and propagation, and the detection of resistance by the NA inhibition assay requires confirmation by sequencing of the NA gene segment to identify the markers of resistance and their presence in the original clinical material.Prior to the 2007-2008 influenza season, the frequency of resistance to NAIs had been very low (<0.5%) among field isolates (28, 35, 36). During the 2007-2008 influenza season, seasonal H1N1 viruses resistant to oseltamivir emerged and spread globally (3, 17, 31, 39), and by April of 2009, the majority of the H1N1 viruses were resistant to oseltamivir but sensitive to zanamivir. Of note, nearly all of the 2009 pandemic H1N1 viruses were sensitive to NAIs (8); only sporadic cases of oseltamivir-resistant viruses with the H275Y mutation in the NA gene segment were reported to the WHO, and they were mainly detected following antiviral drug treatment (5, 6, 42). The H275Y mutation is equivalent to the H274Y mutation in the N2 subtype amino acid numbering. Throughout the text, amino acids are described with the N1 numbering, and the corresponding N2 amino acid numbering is shown in parentheses, when it differs from the N1 numbering. Recent reports on the emergence of oseltamivir resistance highlight the need for close monitoring of the susceptibility of the pandemic H1N1 virus to the available drugs (5, 6, 42). Such information is needed to make informed decisions on measures aimed at managing pandemic virus infections.The molecular markers of NAI resistance are type and subtype specific and are also drug specific (1, 23). The H275Y (H274Y) change is the most commonly reported mutation conferring resistance to oseltamivir in the N1 subtype of NA. This change has been reported not only in seasonal H1N1 viruses but also in highly pathogenic H5N1 viruses (13, 22, 23, 31, 33). The H275Y (H274Y) mutation is also known to reduce susceptibility to the investigational NAI peramivir (23). The amino acid replacement N295S (N294S) in N1 has also been shown to reduce susceptibility to oseltamivir and zanamivir (33, 43). In addition, recent studies have demonstrated that mutations in other residues located in and around the NA active site can alter the susceptibilities of viruses to NAIs. For instance, changes at residues V116, I117, E119, Q136, D199 (D198), and I223 (I222) were associated with reduced susceptibility to NAIs in both seasonal and H5N1 viruses (26-28, 30, 32, 39). Moreover, crystal structure studies with the NAs of H1N1 and H5N1 viruses (9, 37) suggested that mutations at amino acids Q136, K150, and D151 (37) may affect susceptibility to oseltamivir and zanamivir, presumably by interfering with the binding of the drug to the NA. Changes at these residues were reported to reduce the susceptibilities to NAIs of viruses with the N1 enzyme (34; CDC, Atlanta, GA, unpublished data).It is important to develop the tools necessary for the rapid detection of NA markers known or suspected of affecting susceptibility to NAIs. Pyrosequencing has previously been shown to provide a rapid and high-throughput method for the detection of molecular markers of drug resistance in seasonal as well as highly pathogenic avian influenza viruses (4, 8, 15, 16, 19, 30, 31, 38).Here we report on the design and validation of pyrosequencing assays for the detection of signature markers in the M2 and NA gene segments of the pandemic H1N1 viruses.