Document Type

Dissertation - Open Access

Award Date


Degree Name

Doctor of Philosophy (PhD)

Department / School

Biology and Microbiology

First Advisor

Feng Li

Second Advisor

Dan Wang


Hemagglutinin-esterase-fusion (HEF), Influenza D virus, Nucleoprotein (NP), Replication, Reverse-genetics system, Thermal and acid stability


Influenza D virus (IDV) was first identified in 2011 from clinically ill pigs in America. In nearly a decade since its discovery, the virus has been detected in multiple animal species in a vast region of the globe and is considered an important cause of concern to animal and human health. IDV utilizes cattle as the primary reservoir. The viral infection can cause mild respiratory disease in cattle and has been indicated as a causative agent of bovine respiratory disease (BRD) complex that is the most common and costly disease affecting the cattle industry. Moreover, outbreaks of IDV in swine and bovine are increasing, and more genetic and serological evidences show that IDV has a potential to adapt to humans. IDV is unique among four types of influenza viruses. The thermal and acid stability of IDV were examined and directly compared with those of influenza A virus (IAV), influenza B virus (IBV), and influenza C virus (ICV). The results of our experiments demonstrated that only IDV had a high residual infectivity (~2.5 log units of 50% tissue culture infective dose ([TCID50]/ml) after a 60-min exposure to 53°C in solution at a neutral pH, and remarkably, IDV retained this infectivity even after exposure to 53°C for 120 min. Furthermore, the data showed that IDV was extremely resistant to inactivation by low pH. After being treated at pH 3.0 for 30 min, IDV lost only approximately 20% of its original infectiousness, while all other types of influenza viruses were completely inactivated. Finally, replacement of the hemagglutinin (HA) and neuraminidase (NA) proteins of a temperature- and acid-sensitive IAV with the hemagglutinin-esterase fusion (HEF) protein of a stable IDV through a reverse genetic system largely rendered the recombinant IAVs resistant to high-temperature and low-pH treatments. Together, these results indicated that the HEF glycoprotein is a primary determinant of the exceptional temperature and acid tolerance of IDV. Further investigation into the viral entry and fusion mechanism mediated by the intrinsically stable HEF protein of IDV may offer novel insights into how the fusion machinery of influenza viruses evolve to achieve acid and thermal stability, which as a result promotes the potential to transmit across mammal species. To better study IDV at the molecular level, a reverse-genetics system (RGS) is urgently needed, but to date, no RGS had been described for IDV. In this study, we rescued the recombinant influenza D/swine/Oklahoma/1314/2011 (D/OK) virus by using a bidirectional seven-plasmid based system and further characterized rescued viruses in terms of growth kinetics, replication stability, and receptor-binding capacity. Our results collectively demonstrated that RGS-derived viruses resembled the parental viruses for these properties, thereby supporting the utility of this RGS to study IDV infection biology. In addition, we developed an IDV minigenome replication assay and identified the E697K mutation in PB1 and the L462F mutation in PB2 that directly affected the activity of the IDV ribonucleoprotein (RNP) complex, resulting in either attenuated or replication-incompetent viruses. Finally, by using the minigenome replication assay, we demonstrated that a single nucleotide polymorphism at position 5 of the 3’ conserved noncoding region in IDV and influenza C virus (ICV) resulted in the inefficient crossrecognition of the heterotypic promoter by the viral RNP complex. In conclusion, we successfully developed a minigenome replication assay and a robust reverse-genetics system that can be used to further study replication, tropism, and pathogenesis of IDV. Based on the sequences of the hemagglutinin-esterase-fusion (HEF) gene, IDV can be classified into three genetic lineages: D/OK-lineage, D/660-lineage and D/Japan-lineage. The D/swine/Oklahoma/1334/2011 (D/OK) and D/Bovine/Oklahoma/660/2013 (D/660) are the representative strains of the D/OK-, and D/660 -lineages, respectively. We found that the replication of the D/OK virus was approximately 2 log10TCID50/ml lower than that of the D660 virus in different cell lines. Interestingly, by using our reverse genetics system, we generated recombinant chimeric D/OK viruses in that one of each genomic segments was replaced with the segment from the D660 virus, and observed that only the replication fitness of the chimeric D/OK virus with the D660 NP segment was significantly increased. Finally, we identified two positions 247 and 381 within the NP protein were key determinants of the replication difference between the D/OK and D660 viruses. Interestingly, theses amino acid changes in the NP had no effect on IDV RNP activity but may affect virus replication in the late stage of the viral life cycles, which warrants further investigation.

Library of Congress Subject Headings

Sendai virus.
Influenza viruses.



Number of Pages



South Dakota State University

Included in

Microbiology Commons



Rights Statement

In Copyright