Erika Keim

Project title: Pandemic Pivot: Understanding Fate, Transport, Disinfection, and Host Dynamics of Environmental Viral Pathogens and Surrogates in the Era of SARS-CoV-2

Degree: PhD | Program: Environmental and Occupational Hygiene (EOHY) | Project type: Thesis/Dissertation
Completed in: 2021 | Faculty advisor: John Meschke


Environmental contamination of pathogenic viruses is a common occurrence and leading to a number of skin, enteric, and respiratory infections in occupational, nosocomial, and community settings. Viruses with environmental transmission routes have varied abilities to survive and persist in environmental mediums to aiding in their transmission to susceptible hosts. With an on-going novel coronavirus pandemic, investigation on the environmental fate, survival, transmission, and containment of environmental viral pathogens has never been a timelier doctoral dissertation pursuit. The overarching goal of this dissertation was to investigate survival, fate, infection, and viral-host dynamics of environmentally transmitted viral pathogens. To do this I conducted four different lines of investigation using a range of viral pathogens and their surrogates. Aim 1. Indirect environmental infection transmission routes including surface and fomite-based transmission are largely misunderstood, in need of additional research to better understand disease dynamics, and implement successful interventions, such as surface disinfection. Surface transmission of viral pathogens- including SARS-CoV-2- is difficult to pinpoint in epidemiological investigations primarily because it is difficult to eliminate other transmission routes such as direct contact, droplet, and aerosol transmission. Therefore, the first aim of this dissertation was to investigate surface survival of coronaviruses and phage based on temperature, relative humidity, background matrix, and initial viral load. We found that Human Coronavirus OC43 (HCoV-OC43), Murine Hepatitis virus and Pseudomonas bacteriophage phi6 (phi6, enveloped) followed previously studied trends with increased die off with increasing temperature and relative humidity on stainless steel in culture. Additionally, we found that the addition of 1% mucin had a preserving effect for phi6, tripling the average time for a one log10 reduction to occur. This research bolsters and collates viral survival information on two coronaviruses and one enveloped phage and has implications for environmental health in the built environment. Aim 2. Chemical disinfection is an effective way to reduce pathogen loads on surfaces. Previous research has shown that hypohalous acids HOCl and HOBr have been used as effective surface disinfectants on an array of different pathogens, but few studies have directly compared HOCl and HOBr in pure solutions. I conducted a direct comparison of HOCl and HOBr disinfectants at pH=5 on ~108 PFUs of MS2 in both suspension tests and dried on stainless steel surfaces. Suspension tests were conducted with 30 and 60 seconds of contact time and 60, 120, 240, and 480 µM HOCl and HOBr concentrations. Increasing concentrations and time led to increasing log10 reduction values of viable MS2. At 240 µM, HOCl and HOBr were able to achieve log10 reduction values of 3.45 ± 0.32 and 4.27 ± 0.67 (±95% CI) with HOBr significantly more effective at MS2 disinfection than HOCl. This was also true at 480 µM, where HOBr yielded a log10 reduction value of >7.42 ± 0.09 compared to HOCl at 6.31 ± 0.51. Disinfection comparison of MS2 dried on stainless steel coupons with 95, 190, 475, 950, and 1400 µM of HOBr or HOCl, found that HOBr was a more effective disinfectant at all concentrations. Additional work on species characterization and stability found that HOCl remained stable and pure for weeks at pH values less than five, while HOBr was less stable and degraded within hours of being made. These results have implications for use of hypohalous agents for surface disinfection to mitigate viral pathogens. Aim 3. Animal research facilities that use recombinant viruses and viral vectors are held to a variety of regulations related to animal care, occupational health, and biosafety. Fecal waste derived from infected animals must be maintained in facilities that are cleaned daily creating ergonomic and biohazardous challenges for workers and the environment. To help address the issue fecal wastewater contaminated with recombinant viruses and viral vectors poses, chemical disinfection using potassium hydroxide and quaternary ammonium-based disinfectants were evaluated against a suite of seeded enveloped and non-enveloped viral pathogens and phages in animal research wastewater. Enveloped viruses phi6 and simian retrovirus-2 were readily inactivated (> 4-log10 reductions) through chemical disinfection. Non-enveloped viruses MS2 and Human Adenovirus-2 (HAdV-2) experienced minimal to no reduction through disinfection (<0.5 log10 reductions). To better understand partitioning of viruses within fecal wastewater, culture-based assessment of MS2 and phi6 revealed overwhelming partitioning into the liquid portion of samples. Using qPCR, HAdV-2 overwhelmingly partitioned into the liquid portion while HCoV-OC43 partitioned to solids. This research helps inform research facilities using recombinant viruses and viral vectors on the efficacy of chemical disinfection of wastewater. Partitioning results also have implications for SARS-CoV-2 wastewater surveillance. Aim 4. Theiler’s Murine Encephalomyelitis virus (TMEV) is an important virus used in biomedical research to induce multiple sclerosis and epilepsy in murine models. While this virus is readily used in biomedical research, there is a paucity of research on natural disease progression from the gut. Additionally, there is a viral-host interaction involving integrating host glutathione (GSH) into the capsids of public health relevant picornaviruses. To better understand host-viral interactions of TMEV, we first needed to determine how it infects the murine gastrointestinal tract and then determine its sensitivity to changes in host GSH. To do this, I developed a series of murine intestinal enteroid cell lines that had variable expression of host GSH while also representing several intestinal cell types. I also tested TMEV GSH sensitivity in monolayer BHK-21 cells by treating cells with a GSH inhibitor, buthionine sulphoximine, prior to infection. This research did not provide evidence for TMEV replication in murine intestinal enteroids and indicated that TMEV is not sensitive to environmental depletion of intracellular GSH in monolayer. Future research should include more global approaches such as RNA-seq and in vivo experiments. This research is far reaching and covers many topics including surface survival of pathogens, disinfection of viruses on surfaces and in wastewater, and viral-host interactions of a murine enteric virus. Work deriving from this research has far-reaching components within environmental public health, microbiology, and occupational health.