Date of Award

5-2013

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Biomedical Sciences

Track

Microbial Pathogenesis, Immunology, and Inflammation

Research Advisor

Richard J. Webby, Ph.D.

Committee

Kui Li, Ph.D. James P. Ryan, Ph.D. Stacey L. Schultz-Cherry, Ph.D. Paul Thomas, Ph.D.

Abstract

The purpose of this study was to determine mechanisms of reassortment of swine influenza viruses and determine if certain reassortant gene combinations can drive evolution and host adaptation in the context of the triple reassortant internal gene (TRIG) cassette. The TRIG contains a unique combination of a human polymerase basic 1 (PB1) gene with avian polymerase basic 2 (PB2) and polymerase acidic (PA) genes. The remaining internal genes, nucleoprotein (NP), matrix (M), and non-structural (NS) are of swine origin. This differs considerably from classical swine lineage viruses which are drifted variants of the 1918 pandemic with all swine origin genes. We hypothesized that the viruses containing the TRIG cassette reassort more frequently because the polymerase gene constellation is more promiscuous in its ability to replicate with novel HA and NA genes. Better understanding of these mechanisms that control reassortment can be applied to identifying high risk viruses that have an increased ability to reassort and potentially zoonotically transmit to humans. The segmented nature of influenza viruses provides an opportunity for gene segment reassortment in which a virus can exchange segments with another virus. Despite the ability to detect these reassortant viruses, there is little understanding of the mechanisms that control or drive this process. The first two aims addressed a barrier in the influenza field by determining what mechanisms restrict reassortment in particular viral genotypes. This can further be applied to predict the likelihood that other viruses may reassort or be restricted from reassorting. Based on epidemiologic data not all viruses have the same ability to reassort, thus giving some viruses particular advantages over others. However, our studies indicated that there are no virologic specific functions that restrict reassortment, but there are trends demonstrating that the TRIG cassette is more promiscuous than the classical swine viruses. Thus the propensity of the TRIG viruses to reassert, as seen in the field, was likely the result of a change in pig farming rather than one particular viral function. We next assessed the viral fitness of human HA and NA reassortants on both of the swine virus backbones. There were no observable differences with the human H3 and N2 on either of the swine backbones when compared to the parental swine backbones. However, we did see some viral fitness attenuation when the polymerase complex genes were reassorted between the TRIG and classical swine viruses, suggesting that viral growth efficiency is altered if the polymerase genes and presumable their interactions are changed. Influenza gene reassortment plays a major role in the evolution of new viruses, however, it may also drive these reassortant viruses to evolve more quickly and adapt to a new host. The final aim of the study is evaluated the role reassortment plays in driving host adaption and influenza evolution. This aim is designed to test a hypothesis derived from natural observations in which avian-like Eurasian swine virus HAs phylogenetically cluster with increased nucleotide changes in viruses containing the TRIG NS. We hypothesized that the TRIG and TRIG NS increase the number of nucleotide mutations in the HA gene resulting in increased viral diversity leading to more rapid host adaptation as observed by viral fitness in vitro. We found that this evolutionary burst was observed when the TRIG NS is rescued on the classical swine virus backbone; however it was not seen in a wholly TRIG virus. It is likely the introduction of a different gene segment that destabilizes the viral genome leading to the observed evolutionary burst rather than the inherent properties of the TRIG NS. However, this does raise the concern that reassortment can lead to more rapid host adaption through genetic diversity which could lead to novel subtypes entering pigs or humans. Of significant concern is that humans are immunologically naïve to most influenza subtypes. These revelations could help direct the influenza research community to focus on these particular rapidly evolving viruses that pose the most significant threat to public and agricultural health.

DOI

10.21007/etd.cghs.2013.0088

Share

COinS