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Browsing by Author "Mayo, Christie, advisor"

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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.
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    Characterizing the genetic evolution of endemic bluetongue virus strains
    (Colorado State University. Libraries, 2019) Kopanke, Jennifer H., author; Mayo, Christie, advisor; Callan, Rob, committee member; Ebel, Greg, committee member; VandeWoude, Sue, committee member
    Bluetongue virus is an arthropod-borne virus that can cause severe disease in susceptible animals. Transmitted by biting midges in the genus Culicoides, the bluetongue virus particle (genus Orbivirus, family Reoviridae) is composed of ten segments of double-stranded RNA enclosed by a bi-layered, icosahedral capsid. While both wild and domestic ruminants are capable of becoming infected with bluetongue virus, sheep are most likely to develop severe disease characterized by systemic vasculitis, edema, and coagulopathy. Due to its relatively unusual genome structure, bluetongue virus (BTV) is able to evolve via several key mechanisms, including via the accumulation of mutations over time, or more rapidly via reassortment of genome segments. Adding to this genetic complexity, bluetongue virus must maintain fitness in two very disparate hosts: the insect vector and the ruminant. While host-switching is widely accepted as an important aspect of bluetongue virus evolution, the specific features of viral adaptation in each host are poorly characterized. Limited field studies and experimental work from other labs have alluded to the presence of these phenomena at work in the evolutionary trajectory of bluetongue virus, but our overall understanding of the factors that drive or constrain this virus's genetic diversification remains incomplete. In recent years, bluetongue virus has caused significant disease outbreaks among ruminants in enzootic regions, such as the U.S., as well as in areas where bluetongue virus was previously considered exotic, such as northern Europe. Various dynamics including vector range expansion, movement of animals, virus evolution through reassortment and mutation, and environmental factors all may have an integral role in the occurrence of these outbreaks. Not only do bluetongue epizootics carry sometimes profound animal health consequences, but they are also associated with significant economic impacts due to production declines, costly efforts to contain disease spread, and trade restrictions. Collectively, our currently limited understanding of bluetongue virus ecology and evolution dramatically hinders our ability to predict and prevent the occurrence of epizootics associated with orbiviruses. As whole genome sequencing approaches have become increasingly available and affordable, these tools provide a uniquely valuable platform for interrogating underlying viral genetic factors associated with bluetongue disease incursions and outbreaks. Coupling applied fieldwork, in vitro, and in vivo studies with sequencing tools and bioinformatics, the work described in this dissertation seeks to address specific knowledge gaps surrounding bluetongue evolution in North America. In particular, we first queried how an alternating-host transmission cycle affects bluetongue's genetic diversity using an in vitro system, where we leveraged whole genome sequencing and measures of population genetics to understand the role of viral mutation during BTV evolution. We found low rates of overall mutation, leading us to consider whether reassortment is a relatively greater contributor to bluetongue's genetic diversity. Once again using an in vitro platform, we investigated reassortment frequency and segment-specific trends between two enzootic bluetongue virus strains. Our work demonstrated that global shifts in segment frequencies emerged across serial passages, possibly representing preferred reassortant segment combinations. However, most viral segments persisted – even if at very low levels – within the overall population from passage to passage. To better characterize these trends, and to understand whether environmental factors such as temperature might affect their occurrence, we introduced these same viruses into Culicoides sonorensis midges – the predominant North American vector of bluetongue virus – and tracked virogenesis and reassortment across time at three different temperatures. Correlating with other studies, we found that higher temperatures were associated with more rapid virogenesis. However, we were surprised to find that one of the two virus strains replicated poorly in midges orally infected with biologically relevant titers, highlighting potential vector-based barriers to reassortment. Finally, we used whole genome sequencing to characterize circulating strains of bluetongue virus present in Colorado ruminants in 2015 and 2018. We found that numerous strains of bluetongue virus were present among sentinel animals, and that many isolates contained signatures of reassortment. Collectively, our findings demonstrate that reassortment among virus strains is a prominent feature of bluetongue viral evolution. Importantly, there appear to be preferred segment combinations that arise following coinfection, but vector-virus interactions seem to play a central role in modulating the ultimate emergence of reassortant viruses. These studies and others promise to improve our understanding of bluetongue's evolution and ecology, ultimately contributing to the development of better predictive models and management strategies to reduce future impacts of bluetongue epizootics.
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    ItemOpen Access
    Detection of bovine respiratory pathogens using real-time PCR and bead-based technologies
    (Colorado State University. Libraries, 2024) Holmes, Joey, author; Pabilonia, Kristy, advisor; Mayo, Christie, advisor; Ehrhart, Nicole, committee member
    The global cattle industry suffers financial losses of $900 million USD annually from infections caused by respiratory pathogens in the bovine respiratory disease complex (BRD). Accurate and timely detection of BRD pathogens provides cattle producers with a diagnosis so they can institute patient care and prevent pathogen spread. We sought to implement Luminex xTAG technology to detect four pathogens that cause BRD - bovine respiratory syncytial virus (BRSV), bovine viral diarrhea virus (BVDV), bovine herpes virus-1 (BHV-1), and Mycoplasma bovis (M. bovis). We compared singleplex real-time polymerase chain reaction (real-time PCR) to a newly developed xTAG testing protocol. Nucleic acids were extracted from 28 bovine lung samples that previously tested positive on PCR for each of the viral pathogens: BRSV (5), BVDV (5), BHV-1 (5), and M. bovis (5). All samples for BRSV and BHV-1 were detected on xTAG with a mean fluorescent index (MFI) well above 10,000 while detection of BVDV is limited to an MFI of 10,000 and M. bovis is detected inconsistently by xTAG. Lungs from six co-infected animals that tested positive for two BRD pathogens were tested on xTAG and real-time PCR side-by-side, revealing similar findings to the single positive lungs where BHV-1 and BRSV targets are more detectable than BVDV and M. bovis. Spiked pools of all pathogens resulted in MFI decreases as the number of pathogens per sample increases. With proper optimization, Luminex xTAG may be utilized in the veterinary diagnostic setting to circumvent issues with multiplex real-time PCR while maintaining high standards of diagnostic testing.