The H274Y mutation in the influenza A/H1N1 neuraminidase active site following oseltamivir phosphate treatment leave virus severely compromised both in vitro and in vivo
Introduction
The neuraminidase inhibitors (NAI) are a new class of anti-influenza agent. Currently there are two NAI's, zanamivir (Relenza) and oseltamivir phosphate (Tamiflu, Ro64-0796, GS4104), in clinical use. The compounds are specific and potent inhibitors of influenza viral neuraminidase (NA) (Kim et al., 1997). Oseltamivir carboxylate, the active metabolite of oseltamivir phosphate, is the first orally active inhibitor of influenza NA (Kim et al., 1997, Mendel et al., 1998, Hayden et al., 1999). This affords ease of administration compared with the inhaled formulation of zanamivir, for which efficacy depends upon correct use of an inhaler delivery system. Oseltamivir carboxylate binds directly to the NA active site. This binding relies partly on hydrophobic interactions of the inhibitor with conserved amino acid residues within the enzyme active site (Kim et al., 1997). One residue within the viral NA active site shown to be important for oseltamivir carboxylate hydrophobic binding is glutamic acid 276. This lies in close proximity to histidine 274 (N2 numbering) (Lentz et al., 1987, Wang et al., 2000b). The implications of their interaction will be discussed here.
As with all antiviral drugs, selection of drug resistant virus in the clinic presents a potential problem; for example, in treatment of Human Immunodeficiency Virus infection where multiple drug resistant viruses emerge. The experience of resistance generation in influenza virus prior to the introduction of NAI was with the M2 channel blockers, amantadine and rimantadine. Drug resistant virus emerges rapidly following one or two passages in vitro in the presence of amantadine and rimantadine or within 48 h of treatment in man. These drug resistant viruses remain highly pathogenic and transmissible (Oxford et al., 1970, Monto and Arden, 1992, Hayden and Couch, 1992, Hayden, 1996, Sidwell and Huffman, 2000). In contrast, selection of drug resistant virus to NAI in vitro is difficult and many passages are required under continual increasing drug pressure (Bantia et al., 2000, Barnett et al., 1999, Blick et al., 1995, McKimm-Breschkin et al., 1996, McKimm-Breschkin, 2000, Tai et al., 1998). Decreased susceptibility of influenza virus to NAI in antiviral assays following in vitro selection can also arise due to mutational changes in the haemagglutinin (HA), but will not be discussed here.
In N1 NA, a histidine to tyrosine substitution at position 274 has been selected in vitro in two strains of influenza H1N1 using oseltamivir carboxylate. A single amino acid change H274Y occurred in the NA of A/WS/33 whereas in A/Texas/36/91 the H274Y mutation arose following an earlier I222V substitution. This existed as a mixed population with wild type. The I222V mutation alone afforded only a twofold change in sensitivity of enzyme to oseltamivir carboxylate, whereas sensitivity of the double mutant was reduced more than 1000-fold (Wang et al., 2000a). To date there are no reported data on NA mutations selected by zanamivir or BCX-1812 (RWJ-270201) in H1N1 influenza virus in vitro. However, mutation H274N, derived in N1 by point mutation, gave resistance to zanamivir but not oseltamivir carboxylate (Wang et al., 2000b).
Consistent with in vitro studies, the emergence of influenza virus resistant to amantadine and rimantadine in a clinical setting occurs with a high incidence (25–38%) (Hayden, 1996, Daly et al., 2000). In contrast, the emergence of resistant virus in adults with naturally acquired influenza infection treated with oseltamivir phosphate has a low incidence (∼1%) (Covington et al., 2000). The features of NA resistance that has arisen during NAI use in man were largely predicted from in vitro selection studies.
Here we describe the full characterisation, in vitro and in vivo, of an oseltamivir resistant virus with a H274Y mutation in the N1 NA that arose during a study of efficacy of oseltamivir phosphate in experimental influenza A/Texas/36/91 (H1N1) infection in adults (Hayden et al., 1999, Gubareva et al., 2001).
Section snippets
Cells and virus
Madin Darby canine kidney (MDCK) cells were obtained from the laboratory of Dr Alan Hay at the National Institute of Medical Research (London, UK). Cells were grown in Eagles minimum essential medium (MEM) supplemented with 10% fetal calf serum (FCS), 2 mM l-glutamine, 1% non-essential amino acids and 100 Units penicillin–100 μg streptomycin.
Influenza A/Texas/36/91 was obtained as a chicken egg allantoic fluid grown virus from Dr Brian Murphy at the National Institute of Allergy and Infectious
Effect of the H274Y mutation on NA activity
Substrate affinity (Km) and enzymatic activity (Vmax) of mutant NA derived from virus isolated during this clinical study (Gubareva et al., 2001) was equivalent to wild type (Table 1), although the inhibition constant (Ki) for oseltamivir carboxylate was markedly increased (400-fold).
Virus growth in MDCK cell monolayers
Comparison of the growth characteristics of wild type and mutant virus were carried out on confluent MDCK monolayers. Mean viral titres (n=4) were determined over time for each virus (Fig. 1).
The mutant virus was
Discussion
These preclinical studies address the possible consequence of drug resistant H1N1 influenza virus that may emerge following use of oseltamivir phosphate in the clinic. The data here describe the further characterisation, both in vitro and in vivo, of a clinically derived influenza virus A/Texas/36/91 carrying a H274Y NA mutation that arose during an experimental influenza challenge and treatment study in man (Gubareva et al., 2001).
Gubareva has drawn attention to the fact that this virus has an
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