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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.

SUBMITTER: Deyde VM

PROVIDER: S-EPMC2826019 | biostudies-literature | 2010 Mar

REPOSITORIES: biostudies-literature

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

Deyde Varough M VM Sheu Tiffany G TG Trujillo A Angelica AA Okomo-Adhiambo Margaret M Garten Rebecca R Klimov Alexander I AI Gubareva Larisa V LV

Antimicrobial agents and chemotherapy 20091222 3

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 pyroseque ...[more]

PMID: 20028826

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Project description:BackgroundA new strain of human H1N1 influenza A viruses was broken out in the April 2009 and caused worldwide pandemic emergency. The present study is trying to estimate a temporal reassortment history of 2009 H1N1 viruses by phylogenetic analysis based on a total 394 sequences of H1N1viruses isolated from swine, human and avian.ResultsPhylogenetic trees of eight gene segments showed that viruses sampled from human formed a well-supported clade, whereas swine and avian lineages were intermixed together. A new divergence swine sublineage containing gene segments of 2009 H1N1 viruses was characterized, which were closely related with swine viruses collected from USA and South Korea during 2004 to 2007 in six segments (PB2, PB1, PA, HA, NP and NS), and to swine viruses isolated from Thailand during 2004 to 2005 in NA and M. Substitution rates were varied drastically among eight segments and the average substitution rate was generally higher in 2009 H1N1 than in swine and human viruses (F2,23 = 5.972, P < 0.01). Similarly, higher dN/dS substitution ratios were identified in 2009 H1N1 than in swine and human viruses except M2 gene (F2, 25 = 3.779, P < 0.05). The ages of 2009 H1N1 viruses were estimated around 0.1 to 0.5 year, while their common ancestors with closest related swine viruses existed between 9.3 and 17.37 years ago.ConclusionOur results implied that at least four reassortments or transmissions probably occurred before 2009 H1N1 viruses. Initial reassortment arose in 1976 and avian-like Eurasian swine viruses emerged. The second transmission happened in Asia and North America between 1988 and 1992, and mostly influenced six segments (PB2, PB1, PA, HA, NP and NS). The third reassortment occurred between North American swine and avian viruses during 1998 to 2000, which involved PB2 and PA segments. Recent reassortments occurred among avian-to-swine reassortant, Eurasian and classical swine viruses during 2004 to 2005. South Korea, Thailand and USA, were identified as locations where reassortments most likely happened. The co-circulation of multiple swine sublineages and special lifestyle in Asia might have facilitated mixing of diverse influenza viruses, leading to generate a novel virus strain.

Project description:Human-to-swine transmission of seasonal influenza viruses has led to sustained human-like influenza viruses circulating in the U.S. swine population. While some reverse zoonotic-origin viruses adapt and become enzootic in swine, nascent reverse zoonoses may result in virus detections that are difficult to classify as "swine-origin" or "human-origin" due to the genetic similarity of circulating viruses. This is the case for human-origin influenza A(H1N1) pandemic 2009 (pdm09) viruses detected in pigs following numerous reverse zoonosis events since the 2009 pandemic. We report the identification of two human infections with A(H1N1)pdm09 viruses originating from swine hosts and classify them as "swine-origin" variant influenza viruses based on phylogenetic analysis and sequence comparison methods. Phylogenetic analyses of viral genomes from two cases revealed these viruses were reassortants containing A(H1N1)pdm09 hemagglutinin (HA) and neuraminidase (NA) genes with genetic combinations derived from the triple reassortant internal gene cassette. Follow-up investigations determined that one individual had direct exposure to swine in the week preceding illness onset, while another did not report swine exposure. The swine-origin A(H1N1) variant cases were resolved by full genome sequence comparison of the variant viruses to swine influenza genomes. However, if reassortment does not result in the acquisition of swine-associated genes and swine virus genomic sequences are not available from the exposure source, future cases may not be discernible. We have developed a pipeline that performs maximum likelihood analyses, a k-mer-based set difference algorithm, and random forest algorithms to identify swine-associated sequences in the hemagglutinin gene to differentiate between human-origin and swine-origin A(H1N1)pdm09 viruses.IMPORTANCE Influenza virus infects a wide range of hosts, resulting in illnesses that vary from asymptomatic cases to severe pneumonia and death. Viral transfer can occur between human and nonhuman hosts, resulting in human and nonhuman origin viruses circulating in novel hosts. In this work, we have identified the first case of a swine-origin influenza A(H1N1)pdm09 virus resulting in a human infection. This shows that these viruses not only circulate in swine hosts, but are continuing to evolve and distinguish themselves from previously circulating human-origin influenza viruses. The development of techniques for distinguishing human-origin and swine-origin viruses are necessary for the continued surveillance of influenza viruses. We show that unique genetic signatures can differentiate circulating swine-associated strains from circulating human-associated strains of influenza A(H1N1)pdm09, and these signatures can be used to enhance surveillance of swine-origin influenza.

Dataset Information

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

Publications

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

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