Browsing by Author "Mathiason, Candace, committee member"

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Characterizing the effects of bluetongue virus coinfection in Culicoides sonornesis
(Colorado State University. Libraries, 2023) Carpenter, Molly Jean, author; Mayo, Christie, advisor; Mathiason, Candace, committee member; Perera, Rushika, committee member; Simpson, Katie, committee member
Bluetongue virus (BTV) is a segmented, double-stranded RNA virus transmitted by Culicoides biting midges. Infection of domestic and wild ruminants with BTV can result in devastating disease and significant economic losses. In concert with climate change, BTV outbreaks have been characterized by an expanding geographical range and incursions of novel serotypes into endemic regions. As a virus with a segmented genome, reassortment between BTV strains may increase genetic diversity which can alter BTV transmission dynamics and generate epizootic events. While factors driving BTV's expansion are poorly understood, reassortment between virus strains may enhance BTV's ability to spread to new regions. The following studies aimed to investigate different facets of BTV coinfection and reassortment in the Culicoides vector including temperature effects, BTV serotype infection titers, and virus coinfection dissemination. While warmer temperatures have been demonstrated to increase virogenesis, temperature effects on reassortment is not known. The first aim was to evaluate how temperature affects Culicoides survivorship, virogenesis, and progeny virus genotype outcomes in BTV coinfected Culicoides sonorensis. C. sonorensis were provided bloodmeals containing BTV serotype 10 (BTV-10), BTV serotype 17 (BTV-17), or both BTV serotypes and maintained at different temperatures (20°C, 25°C, or 30°C). Every other day, C. sonorensis were collected and processed for BTV qRT-PCR to track virogenesis over time. Co-infected C. sonorensis collected were processed for BTV plaque-isolation (a technique to visualize replicating virus units). The complete genotypes of isolated plaque progeny were determined using shotgun next-generation sequencing. Results indicate that C. sonorensis maintained at warmer temperatures had productive virogenesis earlier in infection than C. sonorenesis held at cooler temperatures. However, C. sonorensis maintained at cooler temperatures had longer mean survival times. Most of the plaque progeny virus genotypes aligned with parental serotype BTV-17, while a few plaques had both parental serotypes represented. While warmer temperatures may accelerate virogensis for earlier potential transmission, there is a trade-off with C. sonorensis mean survival times. Most of the plaque progeny genotypes aligned with BTV-17 indicating that it may be the more fit serotype in the C. sonorensis system. To further explore why the majority of progeny virus aligned with BTV-17, the second aim was to evaluate if different coinfection ratios of BTV-10 and BTV-17 affect progeny virus genotype outcomes. In prior in vitro BTV coinfection and modeling studies, progeny genotypes were dominated by the parental strain with the higher initial multiplicity of infection. To recapitulate this in an in vivo model, C. sonorensis were fed a blood meal containing BTV-10, BTV-17, or both BTV strains with contributing titers of BTV-10 ATCC: BTV-17 at either 90:10, 75:25, 50:50, 25:75, or 10:90 ratios. Pools of five midges were collected in triplicate every other day and processed for pan BTV qRT-PCR to track virogenesis over time. Day ten post-infection midges were collected in pools of ten and processed for plaque isolation and propagation. The complete genotypes of isolated plaques were identified using shotgun next-generation sequencing. Plaque progeny virus genotyping demonstrated an overall trend of progeny virus aligning with the parental serotype with the higher titer. However, a few plaques at a subset of co-infection ratios demonstrated reassortant genotypes with patterns that were suggestive of preferred segment combination. While the parental serotype with the higher contributing titer may have more representation in progeny virus, reassortment events can provide genetic diversity. As reassortment between BTV-10 and BTV-17 was infrequent, it was conjectured that the two parental BTV serotypes did not routinely coinfect the same cells. Thus, the third aim was to determine extent of dissemination and characterize tropism of BTV coinfection in C. sonorensis. In situ hybridization approach was employ using the RNAscope® platform to detect patterns of BTV infection in histologic cross sections of coinfected C. sonorensis. Upon assessment by microscopy, mosaic patterns in which serotypes did not often overlap, suggest that coinfection at the cellular level may not be abundant with these two serotypes. This could be a consequence of superinfection exclusion. Understanding BTV coinfection and its biological consequences will add an important dimension to the modeling of viral evolution and emergence.
Open Access
The role of plants as an environmental reservoir of chronic wasting disease prions
(Colorado State University. Libraries, 2016) Ortega, Aimee Elise, author; Zabel, Mark, advisor; Mathiason, Candace, committee member; Leach, Jan, committee member; Wilusz, Jeffrey, committee member
Transmissible Spongiform Encephalopathies (TSEs) are a group of diseases caused by an abnormal version, PrPRES, of the normal cellular host protein prion protein (Prnp) termed PrPC. Disease is fatal resulting in amyloid deposits and spongiform degeneration in the brain in most but not all cases. Clinical signs can include wasting, increases in salivation, and general motor impairment but many other clinical signs exist and can vary between TSEs. PrPRES is incredibly resistant to inactivation and can withstand radiation, formalin treatment, and autoclaving to name a few tried decontamination methods whereas PrPC is degraded normally. This difference in degradation allows for differentiation between the two protein forms as PrPRES is resistant to degradation by Proteinase K. In the early 1980s this abnormal protein was discovered to be the sole causative agent of the various TSEs which at the time was a novel finding and a novel method of disease transmission. It is thought that slightly misfolded forms of PrPC occur which can then misfold further eventually forming PrPRES. PrPRES then has the ability to act as a template for conversion, converting PrPC. Numerous TSEs exist that affect both humans and a variety of animals. One of the animal TSEs is Chronic Wasting Disease (CWD) which affects cervids such as elk, deer, and moose (Cervus candensis, Odocoileus hemionus, Alces alces) and has become endemic in both free-ranging and captive herds. The exact mechanisms behind spread of CWD are unknown but research has shown that environmental reservoirs play a role in transmission dynamics. We chose to explore whether PrPRES can be detected on or inside grasses and plants naturally exposed to prions in CWD endemic areas by use of Protein Misfolding Cyclic Amplification (PMCA). Here we present novel environmental evidence showing that PrPRES can be found on the surface of multiple plants from Rocky Mountain National Park and mice inoculated with these samples are showing clinical signs of disease.
Open Access
Transmission and shedding of Middle East respiratory syndrome coronavirus from dromedary camels, alpacas, and domestic livestock
(Colorado State University. Libraries, 2016) Adney, Danielle, author; Bowen, Richard, advisor; Mathiason, Candace, committee member; Olea-Popelka, Francisco, committee member; Zabel, Mark, committee member
The Middle East respiratory syndrome coronavirus (MERS-CoV) is a novel betacoronavirus discovered in 2012 that causes severe respiratory disease that can result in death in infected humans. Human-to-human transmission can occur, although zoonotic transmission from dromedary camels plays an important role in transmission. A large percentage of dromedaries in the Middle East and Africa have antibodies specific for MERS-CoV, indicating widespread transmission among camels. In vitro studies indicate that other livestock may be susceptible to infection and transmit virus, however, field studies have not detected any seropositive goats, horses, sheep, or horses. Due to the requirement for specific high-containment facilities research on the role of dromedaries has primarily been limited to field surveys. Here we report experimental infection of dromedary camels with a human isolate of MERS-CoV. The objectives of this study were to characterize clinical disease, shedding, and tissue burdens in infected animals. Experimental infection with the virus resulted in a mild, transient upper respiratory tract infection accompanied by shedding of large amounts of infectious virus. While infectious virus was only detected for a short time viral RNA was detected for much longer, indicating that field studies only using PCR may not be sampling animals able to transmit virus. Due to their cost, size, and temperament, dromedaries are not conducive to high-containment studies, and we hypothesized that alpacas may be a suitable replacement for some studies. This dissertation reports shedding and transmission of MERS-CoV in experimentally infected alpacas (n = 3) or those infected by direct contact (n = 3). Infectious virus was detected in all experimentally infected animals and in 2 of 3 in-contact animals. All alpacas seroconverted and were rechallenged 70 days after the original infection in order to understand if previous infection results in protective immunity. Experimentally infected animals were protected against reinfection, and those infected by contact were partially protected. Necropsy specimens from immunologically naive animals (n = 3) obtained on day 5 postinfection showed virus in the upper respiratory tract. These data demonstrate efficient virus replication and animal-to-animal transmission and indicate that alpacas might be useful surrogates for camels in laboratory studies. Current understanding of MERS-CoV infection suggests that camels become infected as calves or during transport to slaughterhouses, and we hypothesized that vaccinating naïve animals could decrease viral shedding upon exposure. We vaccinated three dromedary camels and two alpaca with a MERS-CoV S protein subunit vaccine. Vaccinated animals developed neutralizing antibodies to MERS-CoV with titers ranging from <10 to 2560 at week 16. Vaccinated animals, as well as two control camels and two control alpaca were challenged with a human isolate of MERS-CoV and were monitored for clinical signs and viral shedding. Vaccinated animals displayed partial protection associated with low or moderate antibody titers whereas the vaccinated alpaca developed high levels of antibody and were protected against infection following viral challenge. In vitro assays indicate that sheep, goats, and horses are susceptible to infection with MERS-CoV. However, unlike dromedaries and alpacas, infected sheep, goats, and horses all failed to shed more than trivial quantities of virus and are therefore unlikely to serve as a source of transmission to humans or other animals. The research presented in this dissertation provides experimental evidence linking dromedaries to MERS-CoV infection. Here, we present the first experimental infection of dromedary camels with MERS-CoV. We describe clinical disease, viral shedding, organ burden, and seroconversion in these animals. It is hoped that these studies will help shape camel sampling as well as allow for a more complete understanding of current field sampling data. Because of the difficulties associated with dromedary camels, we developed a more tractable model of reservoir infection. We found that alpaca model can be a useful substitution for camels in some studies and as well as demonstrate its strengths and weaknesses in a vaccination study. Finally, the data presented here indicate that sheep, goats, and horses do not shed large amounts of infectious virus after challenge with MERS-CoV. This indicates that these animals do not play an important role in viral transmission and that surveillance and vaccine efforts should focus primarily on dromedary camels.