Date of Award


Document Type


Degree Name

Doctor of Philosophy (PhD)


Biomedical Sciences


Microbiology, Immunology, and Biochemistry

Research Advisor

Richard J. Webby, PhD


Colleen B. Jonsson, PhD; Jason W. Rosch, PhD; Charles J. Russell, PhD; Michael A. Whitt, PhD


Highly Pathogenic Avian Influenza;Influenza A Virus;M42;Splicing


Highly pathogenic avian influenza (HPAI) is a continual threat to the commercial poultry industry. In the past 40 years there have been five outbreaks of HPAI in North America. Rapid responses to these outbreaks have been crucial in preventing HPAI from becoming endemic in North America. The three worst outbreaks of HPAI in North America occurred in 1983-84, 2014-15, and 2021-23. The most recent outbreak was the worst North American HPAI outbreak in recorded history and will likely result in endemicity. Here we described the three worst recorded HPAI outbreaks in North America. The first of these outbreaks occurred in 1983-1984 and resulted in culling of almost 17 million chickens, turkeys, and guinea fowl. We demonstrated that the 1980s Pennsylvania linage was unique regarding their M gene splicing. These viruses were capable of producing messenger RNA 4 (mRNA4), which had direct implications in the emergence of virulent viruses from this lineage. The avirulent viruses expressed mRNA4 at high levels. We demonstrated that there was strong selection for virulent viruses with weaker mRNA4 splice donor sites. On at least three separate and independent occasions these viruses decreased their mRNA4 expression. Two of these incidents resulted in the emergence of virulent isolates. We demonstrate that a combination of mutations in the M gene coupled with a single amino acid change point mutation were necessary for these viruses to emerge as virulent. We then compared the two North American HPAI outbreaks from the Asian originating goose/Guangdong (Gs/GD) lineage. This lineage has invaded North America on two separate occasions. In 2014-15 these viruses came from Russia and into Alaska and down the Pacific coast of the United States and reassorted with circulating viruses in wild birds. This outbreak resulted in culling of 50.5 million commercial birds. However, rapid response and stamping out procedures were essential in preventing endemicity. In 2021 viruses from the Gs/GD lineage invaded North America. Similar to the 2014-15 outbreak, these viruses originated from Eurasia, but differed in the flyway used to enter North America. Once in North America these viruses reassorted with circulating influenza A viruses in wild bird populations. In our study we used viruses isolated from wild birds and characterized the genotypes and phenotypes of these emergent reassortant viruses. We demonstrated that after these viruses acquired North American wild bird gene segments, they became neuroinvasive and highly virulent in mammalian animal models. To date, this outbreak has resulted in culling almost 60 million commercial poultry. Our results determined that these viruses, although virulent in mammals, retained mostly avian characteristics. We then assessed the risk of the 2021-23 HPAI outbreak to the poultry industry, wild bird populations, and public health. Current vaccine strategies against emerging HPAI strains requires generating reverse genetics (RG) entirely from complementary DNA (cDNA). One of the more common systems utilizes eight plasmids where the cDNA of all eight viral genomic segments is cloned into the bidirectional expression vector pHW2000. This vector encodes the human RNA polymerase I promoter in the reverse direction and the human cytomegalovirus RNA polymerase II promoter in the forward direction. Additionally, it has a T7 promoter with no functional role in cloning viral RNA or generating reverse genetics viruses. During the cloning process leaky expression of flu transcripts in the transformed bacteria occurs from the cytomegalovirus and T7 promoters. Leaky expression of deleterious transcripts puts mutational pressure on the cDNA insert and has become a widespread problem in the field. Here, we demonstrate that mutating the T7 promoter improves plasmid stability and transformation. Generation of RG candidate vaccine strains are now easier with improved efficacy and decreased leaky bacterial expression. This improved plasmid known as pPW2000 can now be used to clone cDNA of emergent viruses and for increased response times when reassortants or novel HPAI strains emerge in nature and threaten public health.

Declaration of Authorship

Declaration of Authorship is included in the supplemental files.