MOLECULAR SURVEILLENCE OF BLUETONGUE VIRUS: PREVALENCE, GENOMIC LANDSCAPE, AND IDENTIFICATION OF ADDITIONAL VIRUSES USING METAGENOMICS

Abstract

Bluetongue virus (BTV) is an economically important arbovirus of wild and domestic ruminants with a global distribution. Vectored by Culicoides spp. biting midges, this virus can cause significant disease in susceptible hosts, primarily sheep, which is characterized by systemic vasculitis, edema, and ulceration. The cost of BTV outbreaks is high with losses associated with animal morbidities and mortalities, trade restrictions, and implementation of testing and control measures. Control of BTV is primarily through vector control and vaccination strategies but remains a global issue due to its segmented genome and broad antigenic range.The genome of BTV is comprised of ten segments of double-stranded RNA that can undergo reassortment, or the production of a novel progeny virus consisting of genomic segments from both parental strains. Reassortment between different strains may increase genetic diversity, alter BTV transmission dynamics, and enhance its ability to spread to new regions. Segment 2, encoding for the outer capsid protein, is the primary antigenic protein for BTV and determines the strain, or serotype, of the virus. At present there are over 29 described serotypes of BTV that have limited cross-neutralization activity between serotypes which makes control by vaccination alone challenging due to this broad antigenic rage. As this virus can undergo rapid evolution to change both its antigenic segment and others parts of the genome, active surveillance is needed to understand what serotypes circulate endemically in a region and when novel serotypes or genomes are introduced, as these have the potential to cause severe disease. The first aim of this work was to determine the prevalence of BTV in Colorado and surrounding states and which serotypes were circulating over the course of three years, 2021-2023. Using traditional molecular techniques, the prevalence of BTV-positive sheep and cattle was significantly higher in 2021 compared to 2022 and 2023. Additionally, cattle had significantly higher odds of detection compared to sheep. Five different serotypes (BTV-6, -10, -11, -13, and -17) were detected with the predominant serotype that was detected varying annually. These results highlight the ongoing transmission of multiple BTV serotypes within domestic livestock within the U.S. and provide updated information for which serotypes are present in this area. The methods utilized within the first aim provide insight into the current epidemiological situation of BTV within the region but did not provide further information to the genomes of these viruses that are circulating or if reassortment is occurring within this viral population. With the decreased cost and accessibility of whole-genome sequencing, whole genome characterization of BTV is needed to understand reassortment as it is happening in the field. Utilizing samples collected from a single sheep site across the three years of surveillance performed in the first aim, whole-genome sequencing was applied to further characterize these strains and identify evidence of reassortment. Phylogenetic analysis found evidence of reassortment across all genomic segments relative to segment two while nucleotide-level analysis showed high within-serotype homology for most segments. These findings reinforce that reassortment is common among circulating endemic viruses and can involve any segment. With next generation sequencing becoming the standard by which many viruses are characterized, our laboratory has generated extensive sequencing datasets for both vertebrates and invertebrate hosts of BTV and epizootic hemorrhagic disease virus (EHDV), a related orbivirus, directly from field collected samples. Using metagenomics, samples from wild and domestic ruminants and midges were investigated for the presence of additional viruses. Results from this study identified a variety of viruses that have been previously described and a few sequences that are novel. Recovery of a full genome of a mosquito-transmitted orbivirus, Skunk River virus, from a deer represents the first detection of this virus in a vertebrate host. Detection of bovine hepacivirus from a dairy cow represents the second time this virus has been documented in the U.S. Novel sequences recovered from deer samples include sequences from the genus Coltivirus and genus Narnavirus. Sequences recovered from midges included Black Hawk Lake virus, several partitivirus-like segments, and Totivirus nijyuroku. These findings demonstrate the utility of metagenomics in conjunction with sequencing for virus surveillance for further characterization and the discovery of putatively novel viruses. Together, these findings provide an in-depth look at BTV and the wealth of information that can be obtained from field collected samples. This work provides a contemporary view of how much BTV and what strains were circulating during surveillance, the genomes of these viruses and reassortment as it occurs in nature, and the other viruses these animals may be co-infected with.