Influenza A viruses of swine circulating in the United States during 2009–2014 are susceptible to neuraminidase inhibitors but show lineage-dependent resistance to adamantanes
Introduction
Influenza A viruses of swine (IAV-S) can be zoonotic pathogens. The respiratory tract of swine possesses both types of sialic acid receptors recognized by influenza viruses: avian-like (α-2, 3–linked receptors) and human-like (α-2, 6–linked receptors) (Nelli et al., 2010). Therefore, pigs are permissive to infections with both avian-like and human-like influenza A viruses, and some consider pigs as “mixing vessels” in which gene segments from evolutionarily diverse swine, avian, and human influenza viruses can reassort (Scholtissek et al., 1985, Ito et al., 1998). The first influenza pandemic of the 21st century was caused by 2009 H1N1 influenza virus (H1N1pdm09) which possessed genes from IAV-S (Garten et al., 2009, Vijaykrishna et al., 2010).
Antiviral treatment susceptibility is one of the 10 criteria included in the Influenza Risk Assessment Tool (IRAT) that seeks to assess the potential risk posed by influenza A viruses that currently circulate in animals (Trock et al., 2012). The neuraminidase (NA) and matrix (M) proteins are the targets of the two classes of FDA-approved anti-influenza drugs, NA inhibitors (NAIs) and M2 inhibitors, respectively. Amino acid substitutions in these proteins confer drug resistance (Nguyen et al., 2012, Gu et al., 2013). Phenotypic NAI susceptibility of influenza A viruses is evaluated based on the fold-change in IC50 values (the concentration of drug required to inhibit a standardized amount of NA activity by 50%) compared to that in susceptible virus from the same NA subtype/lineage: “normal inhibition” (⩽10-fold change); “reduced inhibition” (10–100-fold change); “highly reduced inhibition” (⩾100-fold change) (WHO, 2012a).
H1N1, H1N1pdm09, H1N2, and H3N2 are endemic subtypes of IAV-S in the U.S. (Vincent et al., 2014). The evolutionary pattern and ecology of IAV-S circulating in the U.S. differ from those circulating in Europe or Asia (Vincent et al., 2014). Since 1998, most of the IAV-S isolated in the U.S. have possessed a triple-reassortant internal genes (TRIG) constellation (Vincent et al., 2008, Webby et al., 2000). IAV-S with TRIG carry human PB1, avian PB2 and PA genes, and classic swine NP, M, and NS genes; they are also characterized by efficient replication in pigs (Pascua et al., 2012). Since 2005, human HA (from H1 and H3 subtypes) and NA (from N1 and N2 subtypes) segments have reassorted with IAV-S carrying TRIG and established a distinct lineage in IAV-S in the U.S. Notably, IAV-S with these NA genes that share ⩾99% identity at the nucleotide and protein levels with those circulated in humans in the 1990s and early 2000s are still endemic in swine. In 2011, the introduction of the M gene from the pandemic H1N1pdm09 viruses (Eurasian avian lineage) to the H3N2 IAV-S (Ducatez et al., 2011) generated the H3N2 variant viruses (H3N2v), which infect humans and have pandemic potential (Lindstrom et al., 2012, Nelson et al., 2012).
IAV-S have been monitored through active and passive surveillance conducted in the U.S. (Webby et al., 2000, Olsen et al., 2000, Anderson et al., 2013, Corzo et al., 2013, Feng et al., 2014), though antiviral surveillance was not part of the overall efforts. A limited number of H1N1 IAV-S isolated in the U.S. during 2005–2009 were susceptible to NAIs (Stoner et al., 2010). The frequency of amantadine-resistant IAV-S in the U.S. remains elusive.
In this study we addressed this gap by characterizing phenotypic and genotypic susceptibility of IAV-S circulating in the U.S. (2009–2011) to NAIs and amantadine. Additionally, we determined the frequency of drug-resistant markers among IAV-S in the U.S. (1930–2014) by screening NA- and M-sequence data available in the influenza research database (IRD) (Squires et al., 2012). We also applied the phylogenetic approach to elucidate the origins of the M gene of IAV-S and assess the distribution of amino acid substitutions associated with antiviral resistance among different lineages.
Section snippets
Viruses and cells
Nasal swabs were collected from pigs at 33 farms during active surveillance from June 2009 to December 2011, in Iowa, Illinois, Indiana, and Minnesota. IAV-S were isolated from nasal swabs by inoculation of Madin–Darby canine kidney (MDCK) cells (ATCC, Manassas, VA) (Corzo et al., 2013). The 105 IAV-S were randomly selected for phenotypic NAI-susceptibility testing and for NA- and M-gene sequencing. (H1N1, 15 strains; H1N1pdm09, 17 strains; H1N2, 62 strains; and H3N2, 11 strains).
Susceptibility to NAIs
Stocks of
Phenotypic susceptibility of IAV-S to NAIs
The NAI susceptibility of 105 IAV-S of 4 HA/NA subtypes are shown in Table 1. N1 and N2 IAV-S displayed normal inhibition by oseltamivir, zanamivir, and peramivir (IC50-fold increase <10 when compared with N1 and N2 reference human influenza viruses). Of interest, IC50 values of 3 H1N1 IAV-S with the I117V-NA were on average 7.3-fold higher for oseltamivir than those of the susceptible control (individual IC50 values are shown in Table 2). NAI susceptibility over the 3-year study remained
Discussion
Given the expanding diversity of IAV-S, both geographically and genetically, and the risk of their role in the genesis of pandemic influenza viruses, it is of concern that so little information is available about the frequency of drug-resistant variants circulating in pigs. To address this question, we used two approaches. First, we applied phenotypic and genotypic methods to examine the susceptibility of IAV-S that have circulated in the U.S. to FDA-approved drugs. Second, we screened NA- and
Conflict of interest
The authors have no personal or financial affiliation with a commercial entity that might pose a conflict of interest.
Acknowledgements
This work was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health, under contract numbers HHSN266200700005C and HHSN272201400006C and by ALSAC. The authors thank Jianling Armstrong, Jeri Carol Crumpton, Adam Rubrum, and Kristi Ann Prevost for technical support and Angela J. McArthur for scientific editing the manuscript. The NAIs oseltamivir carboxylate (oseltamivir) and zanamivir were provided by Hoffmann-La Roche, Ltd. (Basel,
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