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http://www.plospathogens.org/article...l.ppat.1000043
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Human-Like Receptor Specificity Does Not Affect the Neuraminidase-Inhibitor Susceptibility of H5N1 Influenza Viruses
Natalia A. Ilyushina<sup>1</sup><sup>,</sup><sup>2</sup>, Elena A. Govorkova<sup>1</sup>, Thomas E. Gray<sup>3</sup>, Nicolai V. Bovin<sup>4</sup>, Robert G. Webster<sup>1</sup><sup>,</sup><sup>5</sup><sup>*</sup>
1 Division of Virology, Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, Tennessee, United States of America2 Laboratory of Virus Physiology, The D.I. Ivanovsky Institute of Virology RAMS, Moscow, Russia3 Laboratory of Molecular Carcinogenesis, National Institutes of Environmental Health Sciences, Research Triangle Park, North Carolina, United States of America4 Laboratory of Carbohydrate Chemistry, Shemyakin Institute of Bioorganic Chemistry, Moscow, Russia5 Department of Pathology, University of Tennessee Health Science Center, Memphis, Tennessee, United States of America
Abstract
If highly pathogenic H5N1 influenza viruses acquire affinity for human rather than avian respiratory epithelium, will their susceptibility to neuraminidase (NA) inhibitors (the likely first line of defense against an influenza pandemic) change as well? Adequate pandemic preparedness requires that this question be answered. We generated and tested 31 recombinants of A/Vietnam/1203/04 (H5N1) influenza virus carrying single, double, or triple mutations located within or near the receptor binding site in the hemagglutinin (HA) glycoprotein that alter H5 HA binding affinity or specificity. To gain insight into how combinations of HA and NA mutations can affect the sensitivity of H5N1 virus to NA inhibitors, we also rescued viruses carrying the HA changes together with the H274Y NA substitution, which was reported to confer resistance to the NA inhibitor oseltamivir. Twenty viruses were genetically stable. The triple N158S/Q226L/N248D HA mutation (which eliminates a glycosylation site at position 158) caused a switch from avian to human receptor specificity. In cultures of differentiated human airway epithelial (NHBE) cells, which provide an ex vivo model that recapitulates the receptors in the human respiratory tract, none of the HA-mutant recombinants showed reduced susceptibility to antiviral drugs (oseltamivir or zanamivir). This finding was consistent with the results of NA enzyme inhibition assay, which appears to predict influenza virus susceptibility in vivo. Therefore, acquisition of human-like receptor specificity does not affect susceptibility to NA inhibitors. Sequence analysis of the NA gene alone, rather than analysis of both the NA and HA genes, and phenotypic assays in NHBE cells are likely to adequately identify drug-resistant H5N1 variants isolated from humans during an outbreak.
Author Summary
If the avian influenza H5N1 viruses adapt to human hosts, the first step is likely to be a switch in the preference of their viral hemagglutinin (HA) glycoprotein to bind to human rather than avian cell receptors. Such a switch may also alter virus susceptibility to neuraminidase (NA) inhibitors, which are anti-influenza drugs that are likely to be the first line of defense against a pandemic. We generated recombinant A/Vietnam/1203/04-like (H5N1) viruses carrying HA mutations previously shown to alter receptor specificity or affinity. We also discovered a previously unknown route (three simultaneous HA amino acid substitutions) by which highly pathogenic H5N1 viruses can adapt to human receptors. We then used a novel cell-culture?based system (differentiated human airway epithelial NHBE cells) to evaluate the recombinant viruses' resistance to NA inhibitors. None of the HA-mutant recombinants showed reduced drug susceptibility. Our results indicate that the tested HA mutations are unlikely to cause resistance to NA inhibitors in vivo. The NHBE system meets the need for an appropriate cell-culture?based system for phenotypic characterization of drug resistance.
Introduction
The spread of highly pathogenic avian influenza A (H5N1) viruses from Asia to the Middle East, Europe, and Africa raises serious concern about a potential human pandemic [1],[2]. H5N1 avian influenza virus has been reported in poultry in 63 countries; 359 human cases have been confirmed in 14 countries, with a mortality rate >60% [3]. A poor fit between avian viruses and human cellular receptors is thought to be one of the main barriers to efficient transmission of H5N1 influenza viruses between humans [2], [4]?[6]. The hemagglutinin (HA) glycoproteins of avian influenza viruses bind to avian cell-surface receptors whose saccharides terminate in sialic acid (SA)-α2,3-galactose (SAα2,3Gal), whereas those of human influenza viruses bind to human receptors whose saccharides end in SAα2,6Gal. A change in receptor specificity from SAα2,3Gal to SAα2,6Gal is thought to be necessary before avian influenza viruses can cause a pandemic [4]?[6].
Neuraminidase (NA) inhibitors (oseltamivir and zanamivir) are anti-influenza drugs that are likely to be the first line of defense in the event of an influenza pandemic, before antigenically matched influenza vaccine is available [1], [7]?[10]. Although HA mutations that alter viral receptor affinity/specificity can contribute to NA inhibitor resistance in vitro by allowing efficient virus release from infected cells without the need for significant NA activity [9], [11]?[18], the importance of HA mutations in the clinical management of influenza in humans remains uncertain [11], [19]?[23]. One important problem is the lack of a reliable experimental approach (i.e., an appropriate cell-culture?based system) for screening viral isolates for drug sensitivity [9],[11],[19],[20]. HA mutations can either increase or mask NA inhibitor resistance in the available assay systems, which are therefore susceptible to false-positive [24],[25] and false-negative [21],[22] results. This problem is likely to reflect a mismatch between human virus receptors and those in available cell-culture systems. The human airway epithelial cells targeted by influenza virus express high concentrations of SAα2,6Gal-containing receptors, which are present at low concentrations in the continuous cell lines used to propagate influenza viruses [9],[11],[19],[20],[26].
To test whether altered receptor-binding properties of the viral HA glycoprotein of highly pathogenic A/Vietnam/1203/04 (H5N1) influenza virus can reduce susceptibility to NA inhibitors in vivo, we generated 31 recombinant viruses carrying amino acid changes within or near the receptor binding site that alter binding affinity or specificity [27]. To evaluate the recombinant viruses' resistance to NA inhibitors, we used, for the first time, a cell-culture?based system that morphologically and functionally recapitulates differentiated human airway epithelial cells ex vivo [28],[29]. Based on our analysis, we propose that the HA mutations would not be expected to mediate resistance of H5N1 viruses to antiviral drugs, oseltamivir or zanamivir.
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Human-Like Receptor Specificity Does Not Affect the Neuraminidase-Inhibitor Susceptibility of H5N1 Influenza Viruses
Natalia A. Ilyushina<sup>1</sup><sup>,</sup><sup>2</sup>, Elena A. Govorkova<sup>1</sup>, Thomas E. Gray<sup>3</sup>, Nicolai V. Bovin<sup>4</sup>, Robert G. Webster<sup>1</sup><sup>,</sup><sup>5</sup><sup>*</sup>
1 Division of Virology, Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, Tennessee, United States of America2 Laboratory of Virus Physiology, The D.I. Ivanovsky Institute of Virology RAMS, Moscow, Russia3 Laboratory of Molecular Carcinogenesis, National Institutes of Environmental Health Sciences, Research Triangle Park, North Carolina, United States of America4 Laboratory of Carbohydrate Chemistry, Shemyakin Institute of Bioorganic Chemistry, Moscow, Russia5 Department of Pathology, University of Tennessee Health Science Center, Memphis, Tennessee, United States of America
Abstract
If highly pathogenic H5N1 influenza viruses acquire affinity for human rather than avian respiratory epithelium, will their susceptibility to neuraminidase (NA) inhibitors (the likely first line of defense against an influenza pandemic) change as well? Adequate pandemic preparedness requires that this question be answered. We generated and tested 31 recombinants of A/Vietnam/1203/04 (H5N1) influenza virus carrying single, double, or triple mutations located within or near the receptor binding site in the hemagglutinin (HA) glycoprotein that alter H5 HA binding affinity or specificity. To gain insight into how combinations of HA and NA mutations can affect the sensitivity of H5N1 virus to NA inhibitors, we also rescued viruses carrying the HA changes together with the H274Y NA substitution, which was reported to confer resistance to the NA inhibitor oseltamivir. Twenty viruses were genetically stable. The triple N158S/Q226L/N248D HA mutation (which eliminates a glycosylation site at position 158) caused a switch from avian to human receptor specificity. In cultures of differentiated human airway epithelial (NHBE) cells, which provide an ex vivo model that recapitulates the receptors in the human respiratory tract, none of the HA-mutant recombinants showed reduced susceptibility to antiviral drugs (oseltamivir or zanamivir). This finding was consistent with the results of NA enzyme inhibition assay, which appears to predict influenza virus susceptibility in vivo. Therefore, acquisition of human-like receptor specificity does not affect susceptibility to NA inhibitors. Sequence analysis of the NA gene alone, rather than analysis of both the NA and HA genes, and phenotypic assays in NHBE cells are likely to adequately identify drug-resistant H5N1 variants isolated from humans during an outbreak.
Author Summary
If the avian influenza H5N1 viruses adapt to human hosts, the first step is likely to be a switch in the preference of their viral hemagglutinin (HA) glycoprotein to bind to human rather than avian cell receptors. Such a switch may also alter virus susceptibility to neuraminidase (NA) inhibitors, which are anti-influenza drugs that are likely to be the first line of defense against a pandemic. We generated recombinant A/Vietnam/1203/04-like (H5N1) viruses carrying HA mutations previously shown to alter receptor specificity or affinity. We also discovered a previously unknown route (three simultaneous HA amino acid substitutions) by which highly pathogenic H5N1 viruses can adapt to human receptors. We then used a novel cell-culture?based system (differentiated human airway epithelial NHBE cells) to evaluate the recombinant viruses' resistance to NA inhibitors. None of the HA-mutant recombinants showed reduced drug susceptibility. Our results indicate that the tested HA mutations are unlikely to cause resistance to NA inhibitors in vivo. The NHBE system meets the need for an appropriate cell-culture?based system for phenotypic characterization of drug resistance.
Introduction
The spread of highly pathogenic avian influenza A (H5N1) viruses from Asia to the Middle East, Europe, and Africa raises serious concern about a potential human pandemic [1],[2]. H5N1 avian influenza virus has been reported in poultry in 63 countries; 359 human cases have been confirmed in 14 countries, with a mortality rate >60% [3]. A poor fit between avian viruses and human cellular receptors is thought to be one of the main barriers to efficient transmission of H5N1 influenza viruses between humans [2], [4]?[6]. The hemagglutinin (HA) glycoproteins of avian influenza viruses bind to avian cell-surface receptors whose saccharides terminate in sialic acid (SA)-α2,3-galactose (SAα2,3Gal), whereas those of human influenza viruses bind to human receptors whose saccharides end in SAα2,6Gal. A change in receptor specificity from SAα2,3Gal to SAα2,6Gal is thought to be necessary before avian influenza viruses can cause a pandemic [4]?[6].
Neuraminidase (NA) inhibitors (oseltamivir and zanamivir) are anti-influenza drugs that are likely to be the first line of defense in the event of an influenza pandemic, before antigenically matched influenza vaccine is available [1], [7]?[10]. Although HA mutations that alter viral receptor affinity/specificity can contribute to NA inhibitor resistance in vitro by allowing efficient virus release from infected cells without the need for significant NA activity [9], [11]?[18], the importance of HA mutations in the clinical management of influenza in humans remains uncertain [11], [19]?[23]. One important problem is the lack of a reliable experimental approach (i.e., an appropriate cell-culture?based system) for screening viral isolates for drug sensitivity [9],[11],[19],[20]. HA mutations can either increase or mask NA inhibitor resistance in the available assay systems, which are therefore susceptible to false-positive [24],[25] and false-negative [21],[22] results. This problem is likely to reflect a mismatch between human virus receptors and those in available cell-culture systems. The human airway epithelial cells targeted by influenza virus express high concentrations of SAα2,6Gal-containing receptors, which are present at low concentrations in the continuous cell lines used to propagate influenza viruses [9],[11],[19],[20],[26].
To test whether altered receptor-binding properties of the viral HA glycoprotein of highly pathogenic A/Vietnam/1203/04 (H5N1) influenza virus can reduce susceptibility to NA inhibitors in vivo, we generated 31 recombinant viruses carrying amino acid changes within or near the receptor binding site that alter binding affinity or specificity [27]. To evaluate the recombinant viruses' resistance to NA inhibitors, we used, for the first time, a cell-culture?based system that morphologically and functionally recapitulates differentiated human airway epithelial cells ex vivo [28],[29]. Based on our analysis, we propose that the HA mutations would not be expected to mediate resistance of H5N1 viruses to antiviral drugs, oseltamivir or zanamivir.
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