Study of influenza virus neuraminidase inhibitors: Efficacy and molecular characterization of resistant variants.

Abstract

Epidemics and pandemics of influenza continue to be major global health threats. Control measures against influenza infections include vaccination and the use of antivirals. One class of antivirals, the neuraminidase inhibitors (NAIs), specifically inhibit viral neuraminidase (NA) enzymatic activity, which is required for the release of viral progeny and subsequent virus spread through the respiratory tract. Zanamivir and oseltamivir are the two available NAIs that are clinically effective for both prophylaxis and treatment of influenza A and B infections. The objectives of this dissertation were to evaluate the NAI efficacy against a highly pathogenic H5N1 influenza virus with pandemic potential, to characterize the NAI resistant influenza variants for their fitness and transmissibility, and to analyze the relevance of conserved NA residues to NAI resistance. We observed that <italic>in vitro</italic> sensitivity to NAIs did not correlate completely with <italic>in vivo</italic> sensitivity, especially for highly pathogenic H5N1 viruses. This may be due to various factors such as replication efficiency, tissue tropism, and immune evasion that contribute to viral virulence in a living host and directly impact the necessary NAI treatment dosage and schedule. We generated NAI-resistant recombinant viruses of homogeneous genetic background but each carrying a different NA mutation at the conserved NA enzymatic site. With these recombinant viruses, we were able to clarify that NAI resistant viruses with different NA mutations may differ substantially in their fitness and transmissibility due to different level of NA functional loss. To use NAIs in family clusters or in nursing homes, monitoring for the possibility of the emergence of transmissible NAI resistant influenza variants should be taken into account. Among the 18 conserved NA residues that interact with NAIs, mutations at a few residues have been associated with NAI resistance in circulating strains. To determine why only limited conserved NA mutations arise, we analyzed the growth and resistance level of eight recombinant viruses each possessing a single NA mutation. We observed that most mutations at those residues can confer resistance to NAIs, but due to severe loss in NA enzymatic activity, only four out of eight recombinant viruses were able to grow in MDCK cells. Our results explain a possible mechanism for the emergence of NAI resistance and support the strategy to design inhibitors as closely related as possible to the natural ligand of the target. In summary, the findings in this dissertation provided more information for the clinical use of NAIs against influenza epidemics and pandemics.