Carpenter, Molly Jean, authorMayo, Christie, advisorMathiason, Candace, committee memberPerera, Rushika, committee memberSimpson, Katie, committee member2023-08-282025-08-282023https://hdl.handle.net/10217/236949Bluetongue 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.born digitaldoctoral dissertationsengCopyright and other restrictions may apply. User is responsible for compliance with all applicable laws. For information about copyright law, please see https://libguides.colostate.edu/copyright.Culicoidesorbiviruswhole genome sequencingevolutionbluetongue virusreassortmentTextEmbargo expires: 08/28/2025.