Assessing replication, transmission and fitness of influenza viruses with reduced susceptibility to neuraminidase inhibitors

Abstract

© 2019 Rubaiyea FarrukeeInfluenza viruses can cause severe respiratory disease and are responsible for significant morbidity and mortality worldwide. Antivirals are valuable for treatment of influenza infections and neuraminidase inhibitors (NAIs) (oseltamivir, peramivir, laninamivir and zanamivir), are the most commonly available influenza antivirals. NAIs work by blocking the enzymatic activity of the viral neuraminidase (NA) protein, however, viral susceptibility to NAIs can be reduced due to amino acid substitutions that arise in the NA protein. The emergence of variants with reduced NAI susceptibility is a major public health concern. Therefore, the studies described in this thesis aimed to understand the biology of variant influenza viruses with amino acid substitutions in the viral NA protein that reduce sensitivity to NAI in vitro, focussing on their impact on drug effectiveness in vivo, viral fitness, and the risk of emergence and spread. First, we aimed to understand how amino acid substitutions that increase IC50 values (in 50% inhibitory capacity of NAIs), impact drug effectiveness in vivo. Utilising a ferret model, we were able to demonstrate a direct correlation between increased IC50 and reduced effectiveness of oseltamivir, the most widely used NAI, in ferrets. The findings from these experiments highlights the need for revising current guidelines pertaining to influenza B variants, and the risk posed by variants with the commonly reported H273Y or D197N NA (B numbering) substitutions. These substitutions were further characterized in recent viral backgrounds and their impact on NA enzyme function, viral replication and transmission was determined. These experiments demonstrated that both substitutions reduced viral fitness, but the H273Y substitution reduced fitness more than the D197N substitution. We also aimed to understand the risk of variants with reduced NAI sensitivity emerging in A(H7N9) viruses, and selected for drug escape variants by serial passaging reassorted N9 viruses in increasing NAI pressure. These experiments revealed that oseltamivir, peramivir and laninamivir selected for variants with reduced NAI susceptibility, but zanamivir did not. Further analysis on the impact of different amino acid substitutions on N9 NAs derived from different viral backgrounds revealed that with each substitution there was a greater loss of enzyme function in the NA from a Yangtze-River lineage virus than from a Pearl-River Delta lineage virus. The same amino acid substitution can have a different fitness impact in different backgrounds. This was observed with the H275Y substitution (N1 numbering), which was more readily accommodated in 2007 A/Brisbane-like seasonal A(H1N1) viruses compared to earlier A(H1N1) viruses due to permissive substitutions, and therefore became widespread in the human population. In order to predict the risk of H275Y becoming widespread in currently circulating A(H1N1)pdm09 viruses, we established a combination of computational and experimental protocols to predict potentially permissive substitutions in a recent A(H1N1)pdm09 virus, and were able to propose a number of candidate substitutions that will need further evaluation. Overall these studies provided important insights into variant influenza viruses with amino acid substitutions in the viral NA protein that reduce sensitivity to NAI in vitro, particularly in regards to their risk of reducing drug effectiveness and becoming widespread. Furthermore, we have established a number of experimental and computational approaches which can be useful for characterizing fitness of variants with reduced susceptibility to novel antivirals entering the market