Influenza virus susceptibility to antiviral drugs : drug susceptibility profiling, whole-genome mutational landscape and selective pressure footprints

Tese de doutoramento, Ciências e Tecnologias da Saúde (Microbiologia), Universidade de Lisboa, Faculdade de Medicina, 2018Antivirals play an important and decisive role in the clinical management of influenza and in the underlying reduction of related morbidity and mortality. The emergence of antiviral resistance, and particularly of transmissible resistance, poses a serious threat to public health as it could render influenza antivirals useless against circulating viruses. This is even more worrying when considering the current paucity of alternative antiviral therapy choices. This PhD research project aimed at disclosing the susceptibility of human influenza viruses circulating in Portugal to nationally approved antivirals, and at improving the knowledge on the evolutionary dynamics underlying the emergence and/or spread of influenza variants resistant or with decreased susceptibility to neuraminidase inhibitor (NAI) drugs. To this end, the project focused on three main areas: antiviral susceptibility testing; whole-genome sequencing; and selective pressure (SP) footprints on human influenza neuraminidase (NA)(NAI target). Antiviral susceptibility testing was performed on human influenza viruses circulating in both community and hospital settings from 2004/2005 to 2012/2013, after establishing a technological platform for comprehensive evaluation of virus susceptibility to M2 protein inhibitors and the NAIs oseltamivir (OS) and zanamivir (ZA) (objective 1). Important findings were made on: the circulation of drug-resistant A(H3N2) (M2 inhibitors) and former seasonal (H1N1) (OS) viruses; the cut-off for potentially clinically relevant sub-populations of drug-resistant virus; a potential novel amino acid substitution conferring slightly decreased susceptibility to ZA (N2 NA) and a novel source for a variant with decreased susceptibility; and, the virus type or subtype specificity of two amino acid substitutions conferring reduced susceptibility to the drug. Overall susceptibility data contributed at a better understanding of the relationship between virus NAI susceptibility phenotype and genotype and of the natural variations in the in vitro NAI susceptibility of circulating viruses over time. The emergence of new drift variants (former seasonal A(H1N1), A(H3N2)), the co-circulation of distinct virus lineages (influenza B) and the increase in OS drug use (A(H1N1)pdm09) were found to potentially play a role in this latter. Influenza viruses exhibiting resistance or decreased susceptibility to OS and/or ZA were further evaluated through whole-genome sequencing to identify and characterize the amino acid substitutions specific of their genome (objective 2). No genetic support was found for the fitter NA H275Y OS resistant former seasonal A(H1N1) viruses, but mutations known to or that based on its structural location or functional impact may play a role in the overall viral fitness, were identified in the genome of single or few viruses resistant or with decreased susceptibility to the drug. Large datasets of full-length NA gene sequences of worldwide circulating viruses were created to estimate the global and site-specific SP acting on influenza NA, particularly on the sites associated with NAI resistance or reduced susceptibility and/or contacting with the drug (objective 3a). Further temporal splitting of NA gene sequences allowed to investigate for the first time the impact of NAI introduction into clinic (1999) and/or its increased use during 2009 A(H1N1) pandemic on the SP acting on NA (objective 3b). Major findings include: the potential role of positive SP (PSP) in the low-level and locally variable spread of NA H275Y OS-resistant A(H1N1)pdm09 viruses that has been observed in the community; a potential risk of spread of a synergistic drug-resistant (H275Y/S247N) or a RI (S247G) variant in A(H1N1)pdm09 subtype and a RI variant (A395E) in B/Victoria lineage (positive diversifying selection); and the potential lack of impact of both NAI introduction into clinic and its increased use during 2009 A(H1N1) pandemic on the global and site-specific SP acting on influenza NA, with the single exception of site 154 of B/YAM-lineage NA (framework active site residue). Overall mapping of site-specific SP across the different NA subtypes or lineages allowed for further identify 7 potential new regions for drug targeting. This project marked the beginning of influenza antiviral susceptibility testing and monitoring activities in Portugal. It not only established the technological capacity and capability required to perform such activities but also generated comprehensive information on the susceptibility of circulating human influenza viruses, essential to contribute to both global and European influenza surveillance on antiviral susceptibility. The project also contributed at finding potential determinants of viral fitness in the genome of influenza virus resistant or with decreased susceptibility to NAIs, based on its location onto the protein structure; and at elucidating the role of PSP in the evolutionary pathways to NAI resistance or reduced susceptibility.Fundação Calouste Gulbenkian, projetos FCG 76676 e SDH49; Administração Central do Sistema de Saúde, I.P. (ACSS), projeto SDH4