Date of Award

5-2012

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Biomedical Sciences

Track

Microbial Pathogenesis, Immunology, and Inflammation

Research Advisor

Robert G. Webster, PhD

Committee

Elena Govorkova, PhD Tony N. Marion, PhD Paul Thomas, PhD Richard Webby, PhD Michael A. Whitt, PhD

Abstract

Neuraminidase (NA) inhibitors including oral oseltamivir and inhaled zanamivir are among the first line of defense against influenza virus infection. Development of resistance to NA inhibitors is a huge drawback for limited options for the control of influenza. During the first decade of NA inhibitor use, the detection rates of resistance to both NA inhibitors had remained low in circulating influenza viruses. However, the 2008~2009 season was marked by a radical increase of prevalence of oseltamvir resistance from <1% to >90% in worldwide surveillance in less than a year. The resistance was solely linked to NA H275Y variants of seasonal H1N1 viruses, and they are referred as the naturally resistant viruses. A big question remains open about what fundamental molecular changes in the seasonal H1N1 viruses led to the surge of the naturally resistant viruses. When this question remained pending, a novel swine-origin H1N1 influenza virus emerged in Mexico at April 2009, soon spread worldwide replacing the seasonal influenza viruses including the naturally resistant viruses, and marked 2009 with the first influenza pandemic of the 21st century. With sustainably increased worldwide use of NA inhibitors especially oral oseltamivir during the pandemic, the oseltamivir-resistant variants carrying H275Y NA mutation were isolated at low incidence from individuals receiving oseltamivir treatment and a few community clusters. In view of the high prevalence of naturally resistant seasonal H1N1 viruses in the immediate preceding season, there was an urgent need to characterize the transmissibility and fitness of oseltamivir-resistant pandemic H1N1/2009 viruses, although the resistance rates have remained low so far.

We first addressed the urgent question about pandemic viruses by investigating the transmissibility of a closely matched pair of pandemic H1N1/2009 clinical isolates, which only differed at the H275Y NA mutation in their genome, in the ferret model. We found that the H275Y NA mutant H1N1/2009 virus was not transmitted efficiently in ferrets via respiratory droplets, while it retained efficient transmission via direct contact. The wild-type H1N1/2009 virus was efficiently transmitted via both routes. The wild-type and the mutant viruses appeared to cause a similar disease course in ferrets without apparent attenuation of clinical signs. In the growth competition in a ferret, the H275Y mutant virus showed less growth capability than the wild-type virus. The NA of the H275Y mutant virus showed reduced substrate-binding affinity and catalytic activity in vitro and delayed initial growth in MDCK and MDCK-SIAT1 cells. These findings may in part explain its less efficient transmission. The fact that the oseltamivir-resistant H1N1/2009 virus retained efficient transmission through direct contact underlines the necessity of continuous monitoring of drug resistance and characterization of more NA inhibitor-resistant variants of the pandemic H1N1 viruses.

We also sought to resolve the pending question about the naturally resistant seasonal H1N1 viruses by investigating the changes of different seasonal H1N1 viruses in terms of NA genetics, NA proteins attributes and virus fitness. We found that during the seasonal H1N1 virus evolution, two genetically diverged lineages of H1N1 viruses were circulating at different times. The NA protein phenotypes of the two lineages were naturally distinct in the levels of protein expression and enzyme affinity, and accordingly, the H275Y NA mutation had differential effects on the NA proteins and virus fitness of the two lineages. The new lineage NA proteins were inherently higher in protein expression and enzyme affinity than the old lineage NA proteins and thus were able to tolerate the negative effects of the H275Y mutation with a marginal loss of enzyme activity. As a result, the H275Y mutant H1N1 viruses of the new lineage had virus fitness equivalent to the wild-type viruses and were able to continue circulating, becoming the naturally resistant viruses. Further study revealed that 4 different amino acid substitutions played different roles in maintaining high protein expression and enzyme affinity of the new lineage NA proteins; the timeline of the sequential acquisition of the 4 substitutions was consistent with the timeline of emergence of the naturally resistant H1N1 viruses. The identified NA tolerance to the H275Y mutation in the naturally resistant seasonal H1N1 viruses also had implication on the virus fitness of the H275Y mutant H1N1/2009 viruses, as well as on the continuing surveillance monitoring of circulating pandemic H1N1 viruses.

Overall, both studies investigated in vitro and in vivo fitness of H275Y mutant H1N1 viruses relative to their respective wild-type viruses, which were circulating in human beings at different times. These studies correlated the viral fitness of the H275Y mutant viruses with the NA tolerance to the H275Y mutation at protein level, and revealed that the NA tolerance to the H275Y mutation was the molecular determinant of fitness of H275Y mutant H1N1 viruses. The studies have implications on surveillance monitoring of the NA inhibitor resistance in circulating influenza viruses, which underlines the necessity of continuous monitoring of drug resistance incidence, as well as potential genetic and phenotypic changes of constantly evolving influenza viruses.

DOI

10.21007/etd.cghs.2012.0074

Comments

Two year embargo expired May 2014