Date of Award

5-2016

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Biomedical Sciences

Track

Microbiology, Immunology, and Biochemistry

Research Advisor

Richard Webby, PhD

Committee

Vickie S. Baselski, PhD, D(ABMM), F(AAM) Stacey Schultz-Cherry, PhD Robert G. Webster, PhD Michael A. Whitt, PhD

Keywords

bovine, emerging, influenza, pH inactivation, reassortment, zoonotic

Abstract

A novel orthomyxovirus was recently identified from pigs, with subsequent work suggesting the natural reservoir being bovine populations. The virus had genome characteristics most similar to influenza C viruses (ICV) but, due to the extent of sequence divergence, was proposed as a new genus, influenza D virus (IDV). Current literature on IDV has largely focused on the agricultural significance of the virus and provided evidence for the agricultural impact via observation of widespread prevalence and pathology in laboratory infected cattle. However, only one study, which identified 1.3% seroprevalence in a small cohort, has addressed the zoonotic potential of IDV to date, despite evidence that the virus can infect multiple mammalian species. Regardless of zoonotic potential, it is clear that IDV have distinct host ranges from ICV but the molecular markers responsible are not known. In this dissertation we assessed the zoonotic potential of D/swine/Oklahoma/1334/2011 (D/OK), a representative IDV, and conducted studies to investigate receptor binding specificity, temperature sensitivity of replication kinetics, and pH of inactivation, all factors known to affect influenza A virus (IAV) host range.

In order to better address zoonotic potential of D/OK we independently verified the high seroprevalence of D/OK in cattle in the US and found evidence of D/OK circulation in this animal population since at least 2003. We also identified 1% seroprevalence in a cohort of older humans who lived in a rural community with likely exposure to cattle. This seropositivity rate was not, however, elevated compared to earlier studies in populations with low exposure to cattle suggesting that the responses measured were not specific. Further analysis of the seropositive sera indeed found that the IDV seroreactivity was most likely due to cross-reactivity of antibodies induced after prior ICV infection. Despite our inability to identify strong serologic support for zoonotic IDV infection, we did show that D/OK was able to replicate and transmit by direct contact in ferrets and that it replicated robustly in differentiated human respiratory cells, both of which are consistent with an ability to replicate in humans for IAV.

We next explored possible mechanisms for the differences in host range of IDV, which has multiple host species, and ICV, which infects primarily humans. Characterization of the HEF proteins of D/OK and a representative ICV demonstrated that D/OK exhibits altered receptor binding specificity and replicates at higher temperatures than ICV although it does bind receptors present in the human respiratory tract. Using virus-like particles with mutant hemagglutinin-esterase fusion (HEF) proteins, we found that the differences in receptor binding of D/OK were at least partially attributable to residues F143, W201, and F256 that line the putative receptor binding pocket. Surprisingly, we also found that, unlike other orthomyxoviruses, the replication of D/OK was not affected by prior incubation at low pH, raising the possibility that its replication might be pH independent.

Reassortment of orthomyxoviruses is a known mechanism of pandemic emergence of IAV and an informal proxy for genus distinction with viruses from distinct genera considered unable to successfully reassort. Contradictory to published data using conventional approaches we found that, using reverse genetics to force reassortment, D/OK genes could complement each of the corresponding genes from ICV and viable reassortants were produced. It is unclear, however, the biologic impact of these reassortments. The answer to this and other aspects of our work will require a resolution to the current US Governments pause on gain-of-function research. This observation does, however, bring into question the validity of classification of a new influenza genus despite IDV exhibiting the phylogenetic and antigenic divergence used to distinguish a novel genus.

Together the evidence described in this study show that D/OK is widespread in cattle and has characteristics consistent with a zoonotic potential, although we were unable to find convincing evidence for such transmission in a small cohort of humans. We did find that D/OK has many features such as host range, receptor usage, sensitivity to pH, and optimal replication that are distinct from ICV and, we propose, supports its classification as a new genus with the orthomyxovirus family. Continued surveillance and investigation of host species barriers is necessary to further address the public health risk presented by this novel virus.

DOI

10.21007/etd.cghs.2016.0405

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