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Bourbon virus (BRBV) is an emerging pathogen that can cause severe and fatal disease in humans. BRBV is vectored by Amblyomma americanum (lone star ticks), which are widely distributed across the central, southern, and eastern United States. Wildlife species are potentially important for the maintenance and transmission of BRBV, but little is known about which species are involved, and what other factors play a role in the exposure to BRBV. To assess the exposure risk to BRBV among wildlife in the St. Louis area, we collected sera from 98 individuals, representing 6 different mammalian species from two locations in St. Louis County: Tyson Research Center (TRC) and WildCare Park (WCP) from fall 2021 to spring 2023. The sera were used in a BRBV neutralization assay to detect neutralizing antibodies and RT-qPCR for viral RNA analysis. We also sampled and compared the abundance of A. americanum ticks at the two locations and modeled which factors influenced BRBV seropositivity across species. In TRC, we observed a high rate of seropositivity in raccoons (Procyon lotor, 23/25), and white-tailed deer (Odocoileus virginianus, 18/27), but a low rate in opossums (Didelphis virginiana, 1/18). Neutralizing antibodies were also detected in sampled TRC bobcats (Lynx rufus, 4/4), coyotes (Canis latrans, 3/3), and a red fox (Vulpes vulpes, 1/1). The virological analysis identified BRBV RNA in one of the coyote serum samples. In contrast to TRC, all sera screened from WCP were negative for BRBV-specific neutralizing antibodies, and significantly fewer ticks were collected at WCP (31) compared to TRC (2,316). Collectively, these findings suggest that BRBV circulates in multiple wildlife species in the St. Louis area and that tick density and host community composition may be important factors in BRBV ecology.

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Emerging tick-borne viruses such as Powassan virus (POWV), Bourbon virus (BRBV), and Heartland virus (HRTV), whilst rare, can cause severe health problems in humans. While limited clinical cases have been reported thus far in Virginia, the presence of tick-borne viruses poses a serious health threat, and the extent of their prevalence in Virginia is unknown. Here, we sought evidence of POWV, BRBV, and HRTV exposure in Virginia via a serological assessment of wildlife and livestock. Wildlife in Virginia were found to be seropositive against POWV (18%), BRBV (8%), and HRTV (5%), with western and northern regions of the state having a higher prevalence. Multiple wildlife species were shown to have been exposed to each virus examined. To a lesser extent, cattle also showed exposure to tick-borne viruses, with seroprevalences of 1%, 1.2%, and 8% detected in cattle against POWV, BRBV, and HRTV, respectively. Cross-reactivity against other known circulating mosquito-borne flaviviruses was ruled out. In conclusion, there is widespread exposure to tick-borne viruses in western and northern Virginia, with exposure to a diverse range of animal populations. Our study provides the first confirmation that HRTV is circulating in the Commonwealth. These findings strengthen the existing evidence of emerging tick-borne viruses in Virginia and highlight the need for public health vigilance to avoid tick bites.

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The recovery of a Haemaphysalis longicornis Neumann (Acari: Ixodidae) tick from a dog in Benton County, Arkansas, in 2018 triggered a significant environmental sampling effort in Hobbs State Park Conservation Area. The objective of the investigation was to assess the tick population density and diversity, as well as identify potential tick-borne pathogens that could pose a risk to public health. During a week-long sampling period in August of 2018, a total of 6,154 ticks were collected, with the majority identified as Amblyomma americanum (L), (Acari: Ixodidae) commonly known as the lone star tick. No H. longicornis ticks were found despite the initial detection of this species in the area. This discrepancy highlights the importance of continued monitoring efforts to understand the dynamics of tick populations and their movements. The investigation also focused on pathogen detection, with ticks being pooled by species, age, and sex before being processed with various bioassays. The results revealed the presence of several tick-borne pathogens, including agents associated with ehrlichiosis (n = 12), tularemia (n = 2), and Bourbon virus (BRBV) disease (n = 1), as well as nonpathogenic rickettsial and anaplasmosis organisms. These findings emphasize the importance of public health messaging to raise awareness of the risks associated with exposure to tick-borne pathogens. Prevention measures, such as wearing protective clothing, using insect repellent, and conducting regular tick checks, should be emphasized to reduce the risk of tick-borne diseases. Continued surveillance efforts and research are also essential to improve our understanding of tick-borne disease epidemiology and develop effective control strategies.

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Heartland (HRTV) and Bourbon (BRBV) viruses are newly identified tick-borne viruses, isolated from serious clinical cases in 2009 and 2014, respectively. Both viruses originated in the lower Midwest United States near the border of Missouri and Kansas, cause similar disease manifestations, and are presumably vectored by the same tick species, Amblyomma americanum Linnaeus (Ixodida: Ixodidae). In this article, we provide a current review of HRTV and BRBV, including the virology, epidemiology, and ecology of the viruses with an emphasis on the tick vector. We touch on current challenges of vector control and surveillance, and we discuss future directions in the study of these emergent pathogens.

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The recent discovery of Bourbon virus (BRBV) put a new focus on the genus of thogotoviruses as zoonotic, tick-transmitted pathogens within the orthomyxovirus family. Since 2014, BRBV has been linked to several human cases in the Midwest United States with severe acute febrile illness and a history of tick bites. The detection of the virus in the Lone Star tick, Amblyomma americanum, and a high sero-prevalence in wild animals suggest widespread circulation of BRBV. Phylogenetic analysis of the viral RNA genome classified BRBV into the subgroup of Dhori-like thogotoviruses. Strikingly, BRBV is apathogenic in mice, contrasting not only with the fatal disease in affected patients but also with the severe disease in mice caused by other members of the thogotovirus genus. To gain insights into this intriguing discrepancy, we will review the molecular biology and pathology of BRBV and its unique position within the thogotovirus genus. Lastly, we will discuss the zoonotic threat posed by this newly discovered pathogen.

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Bourbon virus (BRBV, family Orthomyxoviridae) is a tickborne virus recently detected in the United States (US). BRBV was first identified from a fatal human case in 2014 in Bourbon County, Kansas. Enhanced surveillance in Kansas and Missouri implicated Amblyomma americanum as the primary vector for BRBV. Historically, BRBV was only detected in the lower midwestern US, but since 2020 it has been reported in North Carolina, Virginia, New Jersey, and New York State (NYS). This study aimed to elucidate genetic and phenotypic characteristics of BRBV strains from NYS through whole genome sequencing and the assessment of replication kinetics in mammalian cultures and A. americanum nymphs. Sequence analysis revealed the existence of two divergent BRBV clades circulating in NYS. BRBV NY21-2143 is closely related to the midwestern BRBV strains but has unique substitutions in the glycoprotein. Two other NYS BRBV strains, BRBV NY21-1814 and BRBV NY21-2666, form a distinct clade unique from previously sequenced BRBV strains. Phenotypic diversification was also detected in NYS BRBV strains compared to each other and midwestern BRBV strains, with BRBV NY21-2143 displaying attenuation in rodent-derived cell culture and a fitness advantage in experimentally infected A. americanum. These data suggest genetic and phenotypic diversification of emergent BRBV strains circulating in NYS that could contribute to increased spread of BRBV in the northeastern US.

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Heartland and Bourbon viruses are pathogenic tick-borne viruses putatively transmitted by Amblyomma americanum, an abundant tick species in Missouri. To assess the prevalence of these viruses in ticks, we collected 2778 ticks from eight sampling sites at Tyson Research Center, an environmental field station within St. Louis County and close to the City of St. Louis, from May - July in 2019 and 2021. Ticks were pooled according to life stage and sex, grouped by year and sampling site to create 355 pools and screened by RT-qPCR for Bourbon and Heartland viruses. Overall, 14 (3.9%) and 27 (7.6%) of the pools were positive for Bourbon virus and Heartland virus respectively. In 2019, 11 and 23 pools were positive for Bourbon and Heartland viruses respectively. These positives pools were of males, females and nymphs. In 2021, there were 4 virus positive pools out of which 3 were positive for both viruses and were comprised of females and nymphs. Five out of the 8 sampling sites were positive for at least one virus. This included a site that was positive for both viruses in both years. Detection of these viruses in an area close to a relatively large metropolis presents a greater public health threat than previously thought.

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In July 2019, Bourbon virus RNA was detected in an Amblyomma americanum tick removed from a resident of Long Island, New York, USA. Tick infection and white-tailed deer (Odocoileus virginianus) serosurvey results demonstrate active transmission in New York, especially Suffolk County, emphasizing a need for surveillance anywhere A. americanum ticks are reported.

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As demonstrated by the 2015 Zika virus outbreak in the Americas, emerging and re-emerging arboviruses are public health threats that warrant research investment for the development of effective prophylactics and therapeutics. Many arboviral diseases are underreported, neglected, or of low prevalence, yet they all have the potential to cause outbreaks of local and international concern. Here, we show the production of virus-like particles (VLPs) using a rapid and efficient recombinant vaccinia virus (VACV) expression system for five tick- and mosquito-borne arboviruses: Powassan virus (POWV), Heartland virus (HRTV), severe fever with thrombocytopenia syndrome virus (SFTSV), Bourbon virus (BRBV) and Mayaro virus (MAYV). We detected the expression of arbovirus genes of interest by Western blot and observed the expression of VLPs that resemble native virions under transmission electron microscopy. We were also able to improve the secretion of POWV VLPs by modifying the signal sequence within the capsid gene. This study describes the use of a rapid VACV platform for the production and purification of arbovirus VLPs that can be used as subunit or vectored vaccines, and provides insights into the selection of arbovirus genes for VLP formation and genetic modifications to improve VLP secretion and yield.

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Bourbon virus (BRBV) was first isolated from a blood sample collected from a male patient living in Bourbon county, Kansas, during the spring of 2014. The patient later died due to complications associated with multiorgan failure. Currently, several BRBV infection-caused deaths have been reported in the United States, and misdiagnosed cases are often undercounted. BRBV is a member of the genus Thogotovirus of the Orthomyxoviridae family, and is transmitted through the Lone Star tick, Amblyomma Americanum, in North America. Currently, there are no specific antivirals or vaccinations available to treat or prevent BRBV infection. Several small molecular compounds have been identified to effectively inhibit BRBV infection of in vitro cell cultures at a single- or sub-micromolar level. Favipiravir, an RNA-dependent RNA polymerase inhibitor, prevented the death of Type I interferon receptor knockout mice infected with BRBV infection.

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Bourbon virus (BRBV) was first discovered in 2014 in a fatal human case. Since then it has been detected in the tick Amblyomma americanum in the states of Missouri and Kansas in the United States. Despite the high prevalence of BRBV in ticks in these states, very few human cases have been reported, and the true infection burden of BRBV in the community is unknown. Here, we developed two virus neutralization assays, a vesicular stomatitis virus (VSV)-BRBV pseudotyped rapid assay and a BRBV focus reduction neutralization assay, to assess the seroprevalence of BRBV neutralizing antibodies in human sera collected in 2020 in St. Louis, MO. Of 440 human serum samples tested, three (0.7%) were able to potently neutralize both VSV-BRBV and wild-type BRBV. These findings suggest that human infections with BRBV are more common than previously recognized. IMPORTANCE Since the discovery of the Bourbon virus (BRBV) in 2014, a total of five human cases have been identified, including two fatal cases. BRBV is thought to be transmitted by the lone star tick, which is prevalent in the eastern, southeastern, and midwestern United States. BRBV has been detected in ticks in Missouri and Kansas, and serological evidence suggests that it is also present in North Carolina. However, the true infection burden of BRBV in humans is not known. In the present study, we developed two virus neutralization assays to assess the seroprevalence of BRBV-specific antibodies in human sera collected in 2020 in St. Louis, MO. We found that a small subset of individuals are seropositive for neutralizing antibodies against BRBV. Our data suggest that BRBV infection in humans is more common than previously thought.

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Haemaphysalis longicornis (Neumann, 1901) (Acari: Ixodidae), the Asian longhorned tick, is an invasive tick species present in the USA since at least 2017 and has been detected in one-third of Virginia counties. While this species is associated with the transmission of multiple pathogens in its native geographical range of eastern Asia, little is known about its ability to acquire and transmit pathogens in the USA, specifically those that are transmissible to humans, although from an animal health perspective, it has already been shown to vector Theileria orientalis Ikeda strains. Emerging tick-borne viruses such as Bourbon virus (genus: Thogotovirus) are of concern, as these newly discovered pathogenic agents have caused fatal clinical cases, and little is known about their distribution or enzootic maintenance. This study examined H. longicornis collected within Virginia (from ten counties) for Bourbon and Heartland viruses using PCR methods. All ticks tested negative for Heartland virus via qRT-PCR (S segment target). Bourbon-virus-positive samples were confirmed on two different gene targets and with Sanger sequencing of the PB2 (segment 1) gene. Bourbon virus RNA was detected in one nymphal stage H. longicornis from Patrick County, one nymph from Staunton City, and one larval pool and one adult female tick from Wythe County, Virginia. An additional 100 Amblyomma americanum (Linnaeus 1758; lone star tick) collected at the same Patrick County site revealed one positive nymphal pool, suggesting that Bourbon virus may have spilled over from the native vector, potentially by co-feeding on a shared Bourbon-virus-infected vertebrate host. Blood tested from local harvested deer revealed a 11.1% antibody seroprevalence against Bourbon virus, exposure which further corroborates that this tick-borne virus is circulating in the southwest Virginia region. Through these results, it can be concluded that H. longicornis can carry Bourbon virus and that pathogen spillover may occur from native to invasive tick species.

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Bourbon virus (BRBV) is an emerging tick-borne orthomyxovirus that causes severe febrile illness in humans. There are no specific treatments for BRBV disease currently available. Here, we developed a highly accessible and robust, quantitative fluorescence-based BRBV minigenome (MG) system and applied it to high-throughput antiviral drug screening. We demonstrated that human dihydroorotate dehydrogenase (DHODH) inhibitors, hDHODH-IN-4 and brequinar, efficiently reduced BRBV RNA synthesis, and validated these findings using infectious BRBV in vitro. The DHODH inhibitors also exhibited high potency in inhibiting MG activities of other orthomyxoviruses with emerging zoonotic potential, including bat influenza A virus, swine influenza D virus, and Thogoto virus. Our newly developed MG system is a powerful platform for antiviral drug screening across the Orthomyxoviridae family, enabling rapid development and deployment of antivirals against future emerging orthomyxoviruses.

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Report of a human death and exposure of white-tailed deer to Heartland virus (HRTV) in Georgia, USA, prompted the sampling of questing ticks during 2018-2019 in 26 sites near where seropositive deer were captured and the residence of the human case-patient. We processed 9,294 Amblyomma americanum ticks in pools by virus isolation in Vero E6 cells and reverse transcription PCR. Positive pools underwent whole-genome sequencing. Three pools were positive for HRTV (minimum infection rate 0.46/1,000 ticks) and none for Bourbon virus. Cell cultures confirmed HRTV presence in 2 pools. Genome sequencing, achieved for the 3 HRTV isolates, showed high similarity among samples but marked differences with previously sequenced HRTV isolates. The isolation and genomic characterization of HRTV from A. americanum ticks in Georgia confirm virus presence in the state. Clinicians and public health professionals should be aware of this emerging tickborne pathogen.

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Thogotoviruses are tick-borne arboviruses that comprise a unique genus within the Orthomyxoviridae family. Infections with thogotoviruses primarily cause disease in livestock with occasional reports of human infections suggesting a zoonotic potential. In the past, multiple genetically distinct thogotoviruses were isolated mostly from collected ticks. However, many aspects regarding their phylogenetic relationships, morphological characteristics, and virulence in mammals remain unclear. For the present comparative study, we used a collection of 10 different thogotovirus isolates from different geographic areas. Next-generation sequencing and subsequent phylogenetic analyses revealed a distinct separation of these viruses into two major clades, the Thogoto-like and Dhori-like viruses. Electron microscopy demonstrated a heterogeneous morphology with spherical and filamentous particles being present in virus preparations. To study their pathogenicity, we analyzed the viruses in a small animal model system. In intraperitoneally infected C57BL/6 mice, all isolates showed a tropism for liver, lung, and spleen. Importantly, we did not observe horizontal transmission to uninfected, highly susceptible contact mice. The isolates enormously differed in their capacity to induce disease, ranging from subclinical to fatal outcomes. In vivo multistep passaging experiments of two low-pathogenic isolates showed no increased virulence and sequence analyses of the passaged viruses indicated a high stability of the viral genomes after 10 mouse passages. In summary, our analysis demonstrates the broad genetic and phenotypic variability within the thogotovirus genus. Moreover, thogotoviruses are well adapted to mammals but their horizontal transmission seems to depend on ticks as their vectors. IMPORTANCE Since their discovery over 60 years ago, 15 genetically distinct members of the thogotovirus genus have been isolated. These arboviruses belong to the Orthomyxovirus family and share many features with influenza viruses. However, numerous of these isolates have not been characterized in depth. In the present study, we comparatively analyzed a collection of 10 different thogotovirus isolates to answer basic questions about their phylogenetic relationships, morphology, and pathogenicity in mice. Our results highlight shared and unique characteristics of this diverse genus. Taken together, these observations provide a framework for the phylogenic classification and phenotypic characterization of newly identified thogotovirus isolates that could potentially cause severe human infections as exemplified by the recently reported, fatal Bourbon virus cases in the United States.

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Vector-borne pathogens, such as Bourbon virus (BRBV), Heartland virus (HRTV), West Nile virus (WNV), and Trypanosoma cruzi (TCZ) are a great threat to public health and animal health. We developed a panel of TaqMan real-time PCR assays for pathogen surveillance. PCR targets were selected based on nucleic acid sequences deposited in GenBank. Primers and probes were either designed de novo or selected from publications. The coverages and specificities of the primers and probes were extensively evaluated by performing BLAST searches. Synthetic DNA or RNA fragments (gBlocks) were used as PCR templates in initial assay development and PCR positive controls in subsequent assay validation. For operational efficiency, the same thermocycling profile was used in BRBV, HRTV, and WNV reverse-transcription quantitative PCR (RT-qPCR) assays, and a similar thermocycling profile without the initial reverse-transcription step was used in TCZ qPCR. The assays were optimized by titrating primer and probe concentrations. The analytical sensitivities were 100, 100, 10, and 10 copies of gBlock per reaction for BRBV (Cq = 36.0 ± 0.7), HRTV (Cq = 36.6 ± 0.9), WNV (Cq = 35.5 ± 0.4), and TCZ (Cq = 38.8 ± 0.3), respectively. PCR sensitivities for vector genomic DNA or RNA spiked with gBlock reached 100, 100, 10, and 10 copies per reaction for BRBV, HRTV, WNV, and TCZ, respectively. PCR specificity evaluated against a panel of non-target pathogens showed no significant cross-reactivity. Our BRBV, HRTV, WNV, and TCZ PCR panel could support epidemiologic studies and pathogen surveillance.

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Following the recent discovery of Bourbon virus (BRBV) as a human pathogen, and the isolation of the virus from Amblyomma americanum (L.) collected near the location of a fatal human case, we undertook a series of experiments to assess the laboratory vector competence of this tick species for BRBV. Larval ticks were infected using an immersion technique, and transstadial transmission of virus to the nymphal and then to the adult stages was demonstrated. Transstadially infected nymphs transmitted virus to adult ticks at very high rates during cofeeding, indicating the presence of infectious virus in the saliva of engorging ticks. Vertical transmission by transstadially infected females to their progeny occurred, but at a low rate. Rabbits fed on by infected ticks of all active life stages developed high titers of antibody to the virus, demonstrating host exposure to BRBV antigens/live virus during tick blood feeding. These results demonstrate that A. americanum is a competent vector of BRBV and indicate that cofeeding could be critical for enzootic maintenance.

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Bourbon virus (BRBV) was first isolated from a patient hospitalized at the University of Kansas Hospital in 2014. Since then, several deaths have been reported to be caused by BRBV infection in the Midwest and Southern United States. BRBV is a tick-borne virus that is widely carried by lone star ticks. It belongs to genus Thogotovirus of the Orthomyxoviridae family. Currently, there are no treatments or vaccines available for BRBV or thogotovirus infection caused diseases. In this study, we reconstituted a replicon reporter system, composed of plasmids expressing the RNA-dependent RNA polymerase (RdRP) complex (PA, PB1, and PB2), nucleocapsid (NP) protein, and a reporter gene flanked by the 3' and 5' untranslated region (UTR) of the envelope glycoprotein (GP) genome segment. By using the luciferase reporter, we screened a few small molecule compounds of anti-endonuclease that inhibited the nicking activity by parvovirus B19 (B19V) NS1, as well as FDA-approved drugs targeting the RdRP of influenza virus. Our results demonstrated that myricetin, an anti-B19V NS1 nicking inhibitor, efficiently inhibited the RdRP activity of BRBV and virus replication. The IC50 and EC50 of myricetin are 2.22 and 4.6 µM, respectively, in cells. Myricetin had minimal cytotoxicity in cells, and therefore the therapeutic index of the compound is high. In conclusion, the BRBV replicon system is a useful tool to study viral RNA replication and to develop antivirals, and myricetin may hold promise in treatment of BRBV infected patients.

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Thogotoviruses are important zoonotic viruses infecting a variety of domestic animals, as well as humans. Among these viruses, Bourbon virus (BRBV) is one of the several human-infecting members, which emerged in the US in recent years and caused human deaths. Here, we report the crystal structure of the BRBV envelope glycoprotein in the postfusion conformation. The structure adopts the typical fold of a class III viral fusion protein and displays an extensive positively charged electrostatic potential pattern, which resembles the glycoprotein of Dhori virus and is consistent with our previous predictions. In addition, compared to other previously defined class III viral fusion proteins, the structures of all thogotovirus glycoproteins and homologs are more similar to herpes virus glycoprotein Bs than to the rhabdovirus G proteins. Thus, class III viral fusion proteins are quite diverse in structure, and sub-classes may have developed during evolution.

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