Biology Ph.D. Dissertations


Detection of Influenza A Viruses From Environmental Lake and Pond Ice

Date of Award


Document Type


Degree Name

Doctor of Philosophy (Ph.D.)


Biological Sciences

First Advisor

Scott Rogers, PhD

Second Advisor

George Bullerjahn, PhD (Committee Member)

Third Advisor

Paul Morris, PhD (Committee Member)

Fourth Advisor

John Castello, PhD (Committee Member)

Fifth Advisor

Robert Midden, PhD (Committee Chair)


Environmental ice is an ideal matrix for the long-term protection of organisms due to the limitation of degradative processes. As a result of global climate change, some glaciers and polar ice fields are melting at rapid rates. This process releases viable microorganisms that have been embedded in the ice, sometimes for millions of years. We propose that viral pathogens have adapted to being entrapped in ice, such that they are capable of infecting naïve hosts after melting from the ice. Temporal gene flow, which has been termed genome recycling (Rogers et al., 2004), may allow pathogens to infect large host populations rapidly. Accordingly, we hypothesize that viable influenza A virions are preserved in lake and pond ice. Our main objective was to identify influenza A (H1-H16) from the ice of a few lakes and ponds in Ohio that have high numbers of migratory and local waterfowl visiting the sites. We developed a set of hemagglutinin subtype-specific primers for use in four multiplex RT-PCR reactions. Model studies were developed by seeding environmental lake water samples in vitro with influenza A viruses and subjecting the seeded water to five freeze-thaw cycles at -20°C and -80°C. Results demonstrated that influenza A viruses survive freeze-thaw cycles at both temperatures with a high rate of viability. We then concentrated the meltwater samples from various locations and years by ultracentrifugation and tested for viable influenza A virions using SPF (specifically pathogen free) embryonated chicken egg cultures. Matrix and hemagglutinin genes of the viruses were detected by RT-PCR (reverse transcription-polymerase chain reaction) and sequencing after several passages depending on the starting concentration of the sample. We analyzed the phylogenetic relationships of the viral strains obtained from environmental ice samples with the known strains available in GenBank based on those two different viral genes. These confirmed the presence of specific subtypes of influenza A viruses. The increase in positive RT-PCR amplicons indicated that the viruses were replicating in the eggs. Our results demonstrated that viable and infectious influenza A virions are preserved in environmental lake and pond ice.