Emerging tickborne viruses vectored by Amblyomma americanum (Ixodida: Ixodidae): Heartland and Bourbon viruses

Abstract

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.

Introduction

Over the last 25 years, isolation and identification of re-emerging and novel arboviruses in the United States has become commonplace. West Nile, chikungunya, and Zika viruses are mosquito-borne viruses from the Old World and have accounted for more than a million human infections in North and South America (Pierson and Diamond 2018, Hadfield et al. 2019, Kramer et al. 2019, Cunha et al. 2020, de Lima Cavalcanti et al. 2022). Powassan virus (POWV), a tick-borne pathogen first isolated in 1958 is now re-emerging, and the annual number of POWV disease cases has been steadily increasing, especially since the association of the virus with Ixodes scapularis Say (Ixodida: Ixodidae) (Mclean and Donahue 1959, Telford et al. 1997, El Khoury et al. 2013). Bacterial and parasitic tick-borne pathogens, particularly Borrelia burgdorferi, Anaplasma phagocytophilum, Babesia microti, and Ehrlichia chaffeensis, account for most vector-borne disease cases in the United States and Canada (Eisen et al. 2017, Rosenberg et al. 2018, Bouchard et al. 2019). A smaller number of human infections each year are caused by emerging viruses transmitted by ticks. In addition to POWV, Heartland (HRTV) and Bourbon (BRBV) viruses have recently been reported in the literature in 2012 and 2015, respectively. HRTV was initially isolated from 2 patients in northwestern Missouri (MO) in 2009 (McMullan et al. 2012). BRBV was isolated and identified from serum samples of a Kansas resident that were submitted to the Centers for Disease Control and Prevention (CDC) for a suspected HRTV infection in 2014 (Kosoy et al. 2015). The distribution of HRTV and BRBV superimposes on the distribution of the putative vector, Amblyomma americanum Linnaeus (Ixodida: Ixodidae), the lone star tick. The purpose of this Forum article is to provide a succinct overview of the emergent HRTV and BRBV and to discuss current knowledge gaps and future study directions, focusing on vector biology and ecology.

Virology

HRTV (Bunyavirales: Phenuviridae; Bandavirus) is closely related to Dabie bandavirus (DBV), commonly referred to as severe fever with thrombocytopenia syndrome virus, an important emerging pathogen distributed in China, Southeast Asia, and Japan (Fig. 1A; Yu et al. 2011, Kim et al. 2013, Takahashi et al. 2014, Tran et al. 2019, Lin et al. 2020). Prior to 2019, HRTV and DBV were members of the phlebovirus genus that separated phylogenetically based upon the associated arthropod vector (Casel et al. 2021). Bandaviruses are single-stranded, negative-sense RNA viruses consisting of 3 segments: small (S), medium (M), and large (L). The S segment encodes for a nonstructural protein and the nucleocapsid protein. The M segment encodes 2 structural glycoproteins essential for cell entry, and the L segment encodes the RNA-dependent RNA polymerase (RdRp), necessary for viral replication (Walter and Barr 2011, Spiegel et al. 2016). HRTV is the only known bandavirus in North America that is associated with human disease. The other viruses in the genus are Bhanja, Guertu, Hunter Island, Kismaayo, Lone star, and Razdan bandaviruses (Kokernot et al. 1969, Shah and Work 1969, Lvov et al. 1978, Matsuno et al. 2013, Wang et al. 2014, Shen et al. 2018). Lone star bandavirus (LSV) was originally isolated from an A. americanum nymph collected in Kentucky (KY) in 1966 (Kokernot et al. 1969). Despite sharing the same vector, LSV and HRTV are only 18–41% similar based on amino acid sequence, serologic cross reactivity is minimal, and LSV pathogenicity in humans is poorly understood (Swei et al. 2013, Riemersma and Komar 2015).

BRBV (Bunyavirales: Orthomyxoviridae; Thogotovirus) is closely related to Oz virus (OZV), recently isolated from A. testudinarium ticks in Japan (Fig. 1B) (Ejiri et al. 2018). Thogotoviruses are single-stranded, negative-sense RNA viruses consisting of 6 segments: polymerase basic subunit 2 (PB2), polymerase basic subunit 1 (PB1), polymerase acidic subunit (PA), surface glycoprotein (GP), nucleoprotein (NP), and the matrix protein (M). The PB2, PB1, and PA encode RdRp subunits responsible for replication, the GP modulates virus attachment and fusion, NPs encapsulate the genome and direct translocation to the nucleus, and the M protein facilitates viral assembly and uncoating (Clerx et al. 1983, Portela et al. 1992, Weber et al. 1998, Bai et al. 2019, Fuchs et al. 2022). Though there are at least 15 recognized thogotovirus strains only 2 are considered separate species, Thogoto virus (THOV) and Dhori virus (DHOV) (Haig et al. 1965, Anderson and Casals 1973). THOV and DHOV are not known to share serological cross reactivity, with greater than 30% divergence of structural genes (Lambert et al. 2015). BRBV, OZV, and Batken virus (BKNV) are classified as Dhori-like viruses (Lambert et al. 2015, Ejiri et al. 2018). Most of the thogotoviruses have been isolated from ticks, although BKNV has also been isolated from several species of mosquitoes (Lvov et al. 1974). Two thogtoviruses have been isolated in the United States, BRBV and Aransas Bay virus (ABV). ABV was isolated from the soft tick, Alectorobius (Ornithodoros) capensis Neumann (Ixodida: Argasidae), collected from abandoned brown pelican nests along coastal islands of Texas in 1975 (Yunker et al. 1979). ABV is genetically more similar to THOV than BRBV (Lambert et al. 2015).

Current mechanisms for HRTV and BRBV evolution remain unknown, but extensive work on DBV and THOV lends insight into the main contributors to genetic diversity. Due to differences in transmission and persistence, tickborne viruses tend to evolve at slower rates than those found in mosquitoes, resulting in relatively stable viral populations (Brackney and Armstrong 2016). In addition, because HRTV and BRBV cycle between tick vector and mammalian host, the viruses encounter divergent selective pressures known to constrain viral diversity, as described by studies with other multihost viruses (Woolhouse et al. 2001, Woelk and Holmes 2002, Li et al. 2008, Ciota and Kramer 2010, Dorrbecker et al. 2010, Grubaugh et al. 2016, Spitaels et al. 2019, Zhou and Yu 2021). Despite this, the error prone RdRp and high replication rates can facilitate genetic diversification (Holland et al. 1982). High homology of HRTV and BRBV RdRps with influenza A and B, THOV, and DHOV PB1 and PA suggest similar endonuclease and RNA synthesis activity that contribute to this diversification (Staunton et al. 1989, Leahy et al. 1997, Weber et al. 1998, Walter et al. 2011). While the tick-borne Powassan virus (POWV, Flaviviridae) and Tick-borne encephalitis virus (TBEV, Flaviviridae) demonstrate relative evolutionary stasis, long-term diversification with consequences for virus ecology and transmission has occurred (Bondaryuk et al. 2023, Vogels et al 2023). In addition, the unique capacity for reassortment of the segmented HRTV and BRBV genomes can be a source of rapid genetic change with the potential to significantly influence transmission and disease, as demonstrated with DBV and THOV reassortment in vertebrate and tick vectors (Davies et al. 1987, Jones et al. 1987, Lam et al. 2013, Liu et al. 2016). More comprehensive genetic surveillance of HRTV and BRBV is needed to better characterize diversity, selective pressures, and the potential role of virus evolution in driving emergence.

Despite homology in RdRps, HRTV, and BRBV contain distinct genetic segments important for host association. The M segment for HRTV encodes the glycoproteins Gn and Gc which together modulate host cell entry and viral assembly (Spiegel et al. 2016). The role of the M segment in vector specificity has not explicitly been studied, but tickborne phlebo- and bandaviruses lack a transmembrane domain in the Gn/Gc proteins present in mosquito-borne phleboviruses such as Rift Valley Fever virus (Spiegel et al. 2016, Hulswit et al. 2021). BRBV GPs are also thought to be associated with vector specificity due to the high similarity to insect baculovirus gp49 (Morse et al. 1992). This homology is absent in the GP receptor binding domains of influenza viruses (Morse et al. 1992, Bai et al. 2019). Other than potential invertebrate host association, knowledge of THOV GP is restricted to its role as a haemagglutinin and fusion protein in vertebrates (Portela et al. 1992). Future work with both HRTV and BRBV GPs is important to elucidate mechanisms of viral entry and vector association across tick genera. With few isolations and limited sequence information, little is known about the genetic and phenotypic variability among BRBV and HRTV. Furthermore, while studies with other thogotoviruses and bandaviruses offer insight into the functional significance of individual genes, species-specific molecular tools are needed to identify relevant genetic correlates.

Clinical Disease and Testing

Since the initial discovery of HRTV, more than 60 human cases have been recorded in 14 states (CDC 2023b). Disease manifestation is similar to ehrlichiosis but does not respond to antibiotic treatment. Hallmark symptoms include fever, fatigue, anorexia, thrombocytopenia, and leukopenia. Other commonly reported symptoms include nausea, headache, confusion, arthralgia, and myalgia (McMullan et al. 2012, Pastula et al. 2014, Brault et al. 2018). Fatal infections have been documented but were usually associated with older individuals who were immunocompromised and/or suffering from comorbidities (Muehlenbachs et al. 2014, Pastula et al. 2014, Carlson et al. 2018, Liu et al. 2023). Late stages of disease in these cases were characterized by kidney and respiratory dysfunction and hypotension leading to organ failure. Mild and asymptomatic infections are known to occur, suggesting that the actual number of infections is underreported (Pastula et al. 2014, Brault et al. 2018, Lindsey et al. 2019, Dupuis et al. 2021). To this end, a serosurvey conducted in 2013 in 10 counties of northwest Missouri found 0.9% of 487 blood donors >16 yr of age were seropositive for HRTV neutralizing antibodies (Lindsey et al. 2019).

Of the thogotoviruses, THOV, DHOV, and BRBV are the only known human pathogens (Lambert et al. 2015, Fuchs et al. 2022). Accurate case counts of BRBV infection are currently unavailable. Three clinical cases of BRBV have been described in the literature, one each in KS, MO, and Oklahoma (Kosoy et al. 2015, OKDOH 2015, MDHSS 2018a, Bricker et al. 2019, Roe et al. 2023), of which 2 were fatal. Missouri Department of Health and Senior Services (MODHSS) lists an additional acute case from the southwest corner of the state that made a full recovery (MDHSS 2018a). Similar to HRTV disease, infection with BRBV is characterized by fever, fatigue, nausea, myalgia, arthralgia, thrombocytopenia, and leukopenia (Kosoy et al. 2015, Bricker et al. 2019, CDC 2023a, Roe et al. 2023). Unlike HRTV, a rash was noted in each of the cases with clinical information available. Late stages of fatal infections were characterized by shock, severe cardiac dysfunction, and pulmonary impairments including excess fluid on the lungs and dyspnea (Kosoy et al. 2015, Bricker et al. 2019). In addition to the 2 nonfatal case reports, mild or asymptomatic infection with BRBV has been documented. Results from a serosurvey of 440 St. Louis, MO patients originally submitted for vitamin D testing indicated 0.7% seropositivity (Bamunuarachchi et al. 2022), and MODHSS reported 5 additional cases of BRBV with no known illness associated with infection (MDHSS 2018b).

Treatment of HRTV and BRBV clinical cases is supportive. Currently, vaccines and antivirals are under early phases of investigation. The antivirals favipiravir, currently under consideration for approval in the United States as a treatment for influenza, and ribavirin have been shown to be effective in preventing HRTV disease in hamsters (Westover et al. 2017) and protected interferon defective mice from lethal challenge with BRBV (Bricker et al. 2019). Initial results of a reverse genetics system suggest that knocking out the nonstructural protein gene of HRTV is protective in AG129 mice and may be suitable as a live-attenuated vaccine candidate. Furthermore, the system may be used in pathogenesis studies (Taniguchi et al. 2022). A BRBV replicon reporter system has been developed as a potential tool in screening anti-viral compounds and the study of viral RNA replication (Hao et al. 2020). The study assessed a number of compounds and found myricetin effectively inhibited BRBV replication in vitro without obvious cytotoxicity, warranting future evaluation.

Considering the overlapping symptomologies of most tick-borne infections (especially milder cases and early disease in symptom onset) and lack of commercially available assays, diagnosis of HRTV and BRBV is not straightforward. Co-infections with other tick-borne diseases are common and further complicate the diagnosis. Testing is limited to the CDC and a few public health laboratories. Clinical case confirmation is based on serology or direct detection of virus (viral RNA) from patient samples. Current serologic tests include IgM and IgG microsphere immunoassays for HRTV and plaque reduction neutralization tests (PRNT) for HRTV and BRBV (Lambert et al. 2015, Basile et al. 2021, Dupuis et al. 2023). Reverse transcription-polymerase chain reaction (RT-PCR) and virus isolation techniques are utilized on acute specimens. Novel molecular and serologic reagents and assays have been developed but are not approved for clinical diagnosis (Calvert and Brault 2015, Shelite et al. 2021, Warang et al. 2021, Bamunuarachchi et al. 2022). Development and validation of Clinical Laboratory Improvement Amendments (CLIA)-approved clinical assays require rigorous standards and may be a lengthy process before an assay is used on human samples.. Given the current scarcity of fully characterized samples, that is, number and volume of potential positive controls, serologic assay development for clinical testing is hampered. Limited testing capacity (primarily performed at the CDC and a few state health departments), generalized symptomologies leading to potential misdiagnosis, low disease incidence, and lack of awareness of lesser-known viral pathogens are impediments to timely and accurate case detection and reporting.

Heartland and Bourbon Virus–Vector Associations

Surveillance of Field Collected Ticks

Amblyomma americanum (L.), commonly referred to as the lone star tick, and colloquially known as the turkey tick given the propensity of immature stages to feed on wild turkeys (Meleagris gallopavo) and other gallinaceous birds (Mock et al. 2001), have been implicated as the primary vector of HRTV and BRBV transmission and maintenance (Savage et al. 2013, 2016, 2017, Godsey et al. 2016, 2021). Amblyomma americanum are 3-host ticks. Larvae and nymphs frequently infest small and medium sized mammals in addition to ground dwelling birds. Adults readily feed on larger hosts such as coyotes (Canis latrans) and white-tailed deer (Odocoileus virginianus) (Kollars et al. 2000). The latter being an important host for all life stages of lone star ticks (Paddock and Yabsley 2007). Each active developmental stage will bite humans. In addition to using the ambush strategy employed by I. scapularis, A. americanum also actively hunt by responding to emitted carbon dioxide and vibrations of host movement (Schulze et al. 1997). Besides HRTV and BRBV, A. americanum are vectors or have been found infected with several human and veterinary pathogens including Ehrlichia chaffeensis, E. ewingii, Francisella tularensis, Rickettsia rickettsia, R. parkeri, and Theileria cervi (Waldrup et al. 1992, Goddard and Varela-Stokes 2009, Fritzen et al. 2011). They are additionally associated with red meat allergy (Alpha-Gal Syndrome) and southern tick-associated rash illness (Varela et al. 2004, Commins et al. 2011, Crispell et al. 2019, Mitchell et al. 2020).

In the United States, A. americanum are distributed from southeast South Dakota through central Texas, eastward through the entire Midwest south of the Great Lakes and Gulf Coast, and extending northeast to southern Maine along the Atlantic coast (Fig. 2). In Canada, A. americanum have been reported in British Columbia, Alberta, Manitoba, Ontario, Quebec, Nova Scotia, and Newfoundland (Nelder et al. 2019). Between 1999-2016, the annual number of lone star submissions increased in Ontario, primarily in the southern and eastern portions of the province (Nelder et al. 2019). The current range expansion of A. americanum is well documented (Ginsberg et al. 1991, Springer et al. 2015, Raghavan et al. 2020) and future modeling predicts recolonization of historic ranges and movement northward via climate change (Sonenshine 2018, Molaei et al. 2019, Sagurova et al. 2019, Ogden et al. 2021), vertebrate host population increases (especially deer) and re-introductions (Paddock and Yabsley 2007, Tsao et al. 2021), landscape patterns and land use changes (reforestation, parcelization, suburban expansion) (Diuk-Wasser et al. 2021), and a combination of all factors (Rochlin et al. 2023). Long distance dispersal of immature stages by migratory birds has been hypothesized, but a study in Canada examined 39,000 passerine and near-passerine birds for ticks and no A. americanum were reported (Ogden et al. 2008).

Prior to 2018, evidence of HRTV transmission was confined to the Midwest United States. In 2012, HRTV was detected in 10 pools of nymphal A. americanum collected from 2 sites at or near one of the first identified HRTV cases (Savage et al. 2013). In 2013, 60 HRTV positive tick pools, 53 nymph pools, and 7 adult pools, were detected at the properties of 5 HRTV clinical cases in Northwest MO, including the 2 index cases (Savage et al. 2016). In 2015 and 2016, HRTV was identified in 2 and 5 A. americanum pools, respectively, that were collected in response to surveillance of the first identified human case of BRBV infection in Bourbon County, KS (Savage et al. 2018b, 2018a). Since 2018, HRTV positive field collected A. americanum have been detected in Alabama, Georgia, Illinois, New York (NY), Pennsylvania, and an additional county in MO (St. Louis). (Newman et al. 2020, Tuten et al. 2020, Dupuis et al. 2021, PADOH 2022, Romer et al. 2022, Aziati et al. 2023). In 2022, HRTV was detected in a single A. americanum submitted from New Jersey and tested at TickReport (Amherst, MA), a commercial tick testing laboratory (TickReport 2023). Infection rates varied across all sites, developmental stages, and time. In the Midwest, the maximum likelihood estimate (MLE) of the infection rate (IR) ranged from 0.32 to 1.99/1,000 for nymphal ticks collected in MO, 2012 and 3.29 to 8.62/1,000 adult males in KS, 2015 (Savage et al. 2013, 2018a). The highest reported MLE (7.60–9.46/1,000 ticks) was observed in adult male ticks from 2 sites in IL (Tuten et al. 2020). In the Southeast, MLE ranged from 0.058/1,000 ticks in AL to 1.35/1,000 ticks in GA (Newman et al. 2020, Romer et al. 2022). The nymphal infection rates (0.00-1.1) in Suffolk County, NY was similar to those in the Midwest and Southeast (Dupuis et al. 2021). To date, HRTV has been detected in A. americanum collected or submitted from at least 13 counties in 8 states (Fig. 3). Currently, HRTV has not been documented or reported from any other species of tick.

BRBV was first detected in A. americanum collected at a site associated with the index case in Bourbon County, KS in 2015 (Savage et al. 2018b). The IR for adult Amblyomma americanum during the entire 2015 surveillance period at this site was 0.25/1,000, but a rate of 7.69/1,000 females collected in June was observed. Retrospective testing of A. americanum collected during surveillance at HRTV index case sites in Missouri in 2013 revealed 3 BRBV positive A. americanum pools (Savage et al. 2017). During the entire sampling period of this study, the MLE of the IR was 0.07 and 0.32/1,000 for nymphs and adult males, respectively. In 2019, BRBV was detected in an individual, partially engorged female A. americanum removed from a resident of Suffolk County, NY by a commercial tick testing laboratory (TickReport). Subsequent enhanced tick surveillance targeting A. americanum across multiple sites in Suffolk County in 2021 resulted in BRBV being detected in 5 of 598 pools of unengorged nymphs (n = 9,972) (Dupuis et al. 2023). Within the last 2 yr, BRBV RNA was detected; in a single pool of A. americanum collected from Patrick County, Virginia in 2021 (Cumbie et al. 2022), in A. americanum collected in Monmouth County, New Jersey (Egizi et al. 2023), and reported in A. americanum collected in 2019 and 2021 from St. Louis County, MO (Aziati et al. 2023). Of note, Cumbie et al reported BRBV RNA detection in 4 pools (2 nymph, 1 adult, 1 larvae) of Haemaphysalis longicornis Neumann (Ixodida: Ixodidae), from 3 counties in Virginia (Cumbie et al. 2022). In total, BRBV has been detected in ticks collected from 10 counties in 5 states (Fig. 4). BRBV has not been detected in other tick species, i.e., Dermacentor variabilis (Say) and I. scapularis, collected during investigations in MO and NY (Savage et al. 2017, Dupuis et al. 2023).

Experimental Infection of Amblyomma americanum

Based on clinical case investigations and active tick surveillance incriminating A. americanum as the putative tick vector of HRTV and BRBV, experimental infections were warranted to confirm vector competence. For each virus, a single vector competence study has been reported. For HRTV, Godsey et al. utilized A. americanum ticks derived from engorged females collected at a field site associated with a human infection and a sympatric strain of HRTV isolated from A. americanum ticks collected in Missouri. Results of the study indicated 6% and 17% of larvae and nymphs, respectively, became infected following immersion (Godsey et al. 2016). The transstadial infection rate was 39% from larvae to nymph and 44% from nymph to adult. Co-feeding transmission from infected nymphs to uninfected larvae was demonstrated at a low level. Vertical transmission from infected female to resulting larvae and horizontal transmission as determined by HRTV neutralizing antibodies in donor rabbits were noted (Godsey et al. 2016). Vector competence of A. americanum for HRTV and viral maintenance through numerous routes have thus been established (Godsey et al. 2016).

A. americanum have also been determined to be competent vectors of BRBV. Godsey et al. infected colony derived larvae using two strains of BRBV from MO and KS. Infection rates were shown to be dependent on viral dose. Transstadial transmission from larvae to nymph and nymph to adult was established. BRBV was detected from approximately 23% of adult ticks resulting from donor nymphs co-feeding with infected larvae. Horizontal transmission was evidenced by seroconversion in bloodmeal hosts. The vertical transmission rate was 8% of the 48 larval pools tested. All positive larval pools were from 2 of 4 infected females (Godsey et al. 2021). As evidenced by the limited number of vector competence studies, knowledge of tick and vertebrate host competence is limited. These studies are further constrained by the lack of established animal models for HRTV and BRBV, the availability of regionally relevant tick populations, and the need for reliable artificial blood feeding systems for maintenance and infection of ticks.

Alternative Tick Vectors

Though A. americanum has been incriminated as the primary vector for HRTV and BRBV, experimental infections of H. longicornis and virus (RNA) detection in this species in nature, suggest both HRTV and BRBV may be supported by alternative tick vectors. The first discovered established population of H. longicornis was reported from an infested sheep in New Jersey in 2017 (Rainey et al. 2018). Concerted surveillance efforts within a year of this discovery, documented H. longicornis in 8 additional states (Beard et al. 2018). Following a review of archived tick specimens, it was determined that H. longicornis had been present since 2010 (Beard et al. 2018) and as of September 2022 H. longicornis has been found in 18 states (USDA 2023). Field evidence of vertical transmission has been reported with the closely related DBV and the enzootic vector H. longicornis (Wang et al. 2015), providing the impetus for an experimental infection study of H. longicornus with HRTV. Results revealed high infection rates (100% after microinjection, n = 8) in adults and transovarial transmission rates in larvae (100%, n = 15 pools) (Raney et al. 2022). Bandaviruses, including DBV, have been isolated from several genera of ticks. In addition to H. longicornis, DBV has been isolated from Rh. (Boophilus) microplus Canestrini, the southern cattle fever tick, which was previously eliminated from the United States in the 1940s but recently identified in south Texas (Osbrink et al. 2020, 2022). Bhanja bandavirus has been isolated from Haemaphysalis spp, A. variegatum, Dermacentor marginatus Sulzer, Hyalomma truncatum Koch, and Rh. decoloratus Koch, suggesting a wide vector host range (Hubalek 2009). Hunter Island bandavirus was originally isolated from Ixodes eudyptidis (Maskell), a tick that specializes on sea birds (Wang et al. 2014).

THOV has been isolated from H. longicornis in Japan and vector competence was subsequently established with a Japanese isolate of THOV in this tick species (Yoshii et al. 2015, Talactac et al. 2018). Furthermore, BRBV RNA was detected in multiple tick pools of H. longicornis collected in Virginia (Cumbie et al. 2022), indicative of pathogen spillover. THOV has also been isolated from several species of Rhipicephalus, including R. sanguineus Latreille, A. variegatum, Hy. truncatum, and Hy. anatolicum Koch (Haig et al. 1965, Albanese et al. 1972, Filipe and Calisher 1984, Calisher et al. 1987). Co-feeding transmission has been demonstrated in the laboratory with THOV and Rhipicephalus appendiculatus Fabricius and A. variegatum Fabricius (Jones et al. 1990). DHOV has been isolated from Hyalomma spp., Dermacentor spp., and Amblyomma spp. (Anderson and Casals 1973, Sang et al. 2006a).

Ixodes scapularis and D. variabilis are important disease vectors in the United States and the geographic and vertebrate host ranges for each of these species overlap with A. americanum. There is evidence of co-infestation of A. americanum with I. scapularis on white-tailed deer (Apperson et al. 1990, Allan et al. 2001), an important bloodmeal host with high seroprevalence to HRTV and BRBV in nature, as further discussed later. Recent experimental work performed in our laboratory has demonstrated the ability of BRBV but not HRTV to replicate efficiently in I. scapularis nymphs following immersion. Additional studies to assess transmission (transstadial, vertical, horizontal, and co-feeding) are necessary for classifying vector status. While tick species other than A. americanum, in particular H. longicornus, may not be as important for transmitting HRTV or BRBV to humans, their ability to support either or both viruses could potentiate viral dispersal and emergence in new transmission cycles and foci. Another tick species of concern in the United States is A. maculatum Koch (Ixodida: Ixodidae). Amblyomma maculatum is currently undergoing a north- and west-ward range expansion from the Gulf Coast into the coastal mid-Atlantic, western KY and Tennessee, and westward into Oklahoma and KS (Teel et al. 2010, Alkishe and Peterson 2022). An established population of this species has been reported in CT (Molaei et al. 2021). They feed on a diverse array of vertebrate hosts, including migratory passerines responsible for dispersal (Kinsey et al. 2000, Cohen et al. 2010). Laboratory experiments and evidence from the field established co-feeding transmission of R. parkeri from infected A. maculatum to nymphal A. americanum, providing a model for pathogen exchange between sympatric tick vectors (Wright et al. 2015).

Co-infections of two or more human pathogens found in a single tick have been reported for A. americanum and especially I. scapularis (Schulze et al. 2005, 2013, Tokarz et al. 2010, Aliota et al. 2014, Hersh et al. 2014, Killmaster et al. 2014, Prusinski et al. 2014, Cross et al. 2018, Sanchez-Vicente et al. 2019, Cutler et al. 2021, Milholland et al. 2021, Zembsch et al. 2021, Hart et al. 2022, Schwartz et al. 2022). Elucidating the effects of co-infection of diverse pathogens (viral, bacterial, parasitic) within A. americanum and other species should be included in future studies. Hart et al. investigated the interactions of a virus-bacteria combination in I. scapularis ticks and found increased viral replication and dissemination in ticks experimentally infected with POWV and B. burgdorferi compared to ticks infected with POWV alone (Hart et al. 2022). In addition, replication of B. burgdorferi was not negatively impacted by the presence of POWV. HRTV and BRBV share the same vector, infect the same hosts, and circulate in the same region. In fact, HRTV and BRBV have been isolated from A. americanum collected from study sites in KS and NY during the same sampling campaign (Savage et al. 2018b, Dupuis et al. 2023). Given relatively low overall infection rates, the chance an individual tick is infected with both viruses is currently remote, but increasing prevalence could lead to increasing human disease risk from co-infections. Interactions between pathogens can be neutral (effect of POWV on B. burgdorferi), advantageous for one (effect of B. burgdorferi on POWV), synergistic, or antagonistic and have impacts on pathogen transmission, tick immune response, vertebrate host response, etc. Beyond human pathogens, ticks harbor endosymbionts and other microbiota (Greay et al. 2018, Guizzo et al. 2022a, 2022b), thus further complicating intra-host interactions.

Heartland and Bourbon Virus Antibodies in Nonhuman Vertebrates

During HRTV and BRBV epidemiologic case investigations, A. americanum was incriminated as a potential vector. Due to the wide host range of this tick, researchers targeted wild and domestic animals at or near sites of likely case exposure to assess seroprevalence in these populations. During 2012-2013 in Missouri, 42.6% of northern raccoons (Procyon lotor), 17.4% of horses (Equus ferus caballus), 14.3% of white-tailed deer, 7.7% of dogs (Canis lupus familiaris), and 3.8% of Virginia opposums (Didelphis virginiana) contained specific neutralizing antibodies to HRTV. Eastern cottontails (Sylvilagus floridanus), Fox squirrels (Sciurus niger), cats (Felis catus), and 132 birds of 26 species were all seronegative (Bosco-Lauth et al. 2015). A retrospective serosurvey of banked sera from white-tailed deer (n = 396), raccoons (n = 949), moose (Alces alces, n = 22), and coyotes (Canis latrans, n = 61) from the eastern and southern United States revealed HRTV transmission across wide swaths of the Midwest and Southeast, areas with burgeoning populations of A. americanum. Surprisingly, HRTV seroprevalence was observed in New England (Maine, New Hampshire, Vermont), an area with few, if any, established populations of A. americanum (Springer et al. 2015), suggesting an alternative vector, the circulation of a closely related virus, or frequent dispersal of HRTV-infected A. americanum to these areas. Similarly, a seroprevalence study of domestic and captive farm animals in Minnesota, also a state with no evidence of established populations of A. americanum, revealed antibodies to HRTV or a DBV-like virus (Xing et al. 2013). Overall seroprevalence was 13.9% of deer, 7.0% of raccoons, 18.2% of moose and 18.0% of coyotes. Rates vary by location (Riemersma and Komar 2015). A third study investigated archived deer sera from the southeastern United States collected between 2001-2015. This period includes the time prior to the initial isolation and identification of HRTV in Missouri, 2009. Overall, 7.3% of 783 deer were seropositive for HRTV neutralizing antibodies. Antibody was present every year in the deer sampled except for 2004, suggesting enzootic HRTV transmission at least 8 yr prior to the first recognized disease cases (Clarke et al. 2018b). HRTV seroprevalence was approximately 10.0% in hunter-harvested white-tailed deer sampled from Suffolk County, NY (Dupuis et al. 2021), a location with a robust A. americanum population, yet similar to rates observed for deer in Maine (11.0%) and Vermont (10.0%) (Riemersma and Komar 2015). Counties or areas in the United States where HRTV seropositive animals have been detected are illustrated in Fig. 3.

In northwestern MO, BRBV neutralizing antibodies were detected in white-tailed deer (86.0%), northern raccoons (50.0%), eastern cottontails (22.0%), horses (4.0%), and dogs (15.0%). No antibodies were detected in cats, fox squirrels, or Virginia opossums in this same study (Jackson et al. 2019). During a survey of 32 white-tailed deer in North Carolina, 56% were seropositive to BRBV (Komar et al. 2020). In Virginia, BRBV seropositive animals included white-tailed deer (11.2%), groundhogs (Marmota monax, 60.0%), and northern raccoons (25.0%). Two striped skunks (Mephitis mephitis) and an eastern cottontail were seronegative for BRBV antibodies (Cumbie et al. 2022). Dupuis et al. 2023, demonstrated a 66.5% seroprevalence in white-tailed deer harvested in Suffolk County, NY. Lower rates were observed in western NY (3.8%), central NY (1.2%), and the Hudson Valley region of NY (1.7%). These areas have fewer established populations of A. americanum, especially compared to Long Island (Means and White 1997).

Reservoir Host Competence

Only 2 studies have been published that have examined the role of vertebrates in HRTV transmission. Bosco-Lauth et al. infected common laboratory strain animal models (chickens, rabbits, hamsters, and mice) to assess for potential future tick feeding experiments and or disease model suitability (Bosco-Lauth et al. 2016). Also included in this study were raccoons due to their high seroprevalence rates in nature (Bosco-Lauth et al. 2015, Riemersma and Komar 2015) and goats because of previous serologic investigations and experimental infections of DBV in China (Zhao et al. 2012, Jiao et al. 2015). All animals were needle inoculated with a human derived strain of HRTV. In the study, only interferon receptor-deficient mice (strain Ag129) produced a detectable viremia and exhibited signs of clinical illness. Neutralizing antibodies were observed in raccoons and goats despite a lack of viremia, suggesting these animals became infected but likely are not amplifying hosts for HRTV.

In the second study (Clarke et al. 2018a), white-tailed deer fawns were needle inoculated with HRTV (Missouri, 2009 strain), since deer are frequently fed upon by A. americanum and seroprevalence to HRTV in nature has been observed (Riemersma and Komar 2015, Clarke et al. 2018b, Dupuis et al. 2021). Of 5 fawns acquired for experimentation, 2 had evidence of prior exposure to HRTV. None of the deer became viremic or showed signs of clinical illness, including fever. Viral shedding was not observed, and virus was not isolated from any tissue after necropsy. It was determined white-tailed deer are not a reservoir for HRTV. Deer likely play an important role in A. americanum population maintenance/expansion and a potential host for nonviremic (cofeeding) transmission.

To date, there are no reported studies of reservoir competence for BRBV, but in vivo characterization of 10 thogotovirus isolates was performed utilizing C57BL/6 mice. Unlike the majority of thogotoviruses included in the study, BRBV and the closely related OZV, produced no measurable virus in any tissue/organ measured (brain, liver, lung, kidney, spleen, serum) (Fuchs et al. 2022).

These studies highlight the potential for HRTV and BRBV maintenance independent of a reservoir host. Of note, POWV is similar in that a definitive reservoir host has not been identified in nature and infection rates are relatively low in Ixodes spp. populations. It is hypothesized that transovarial transmission and cofeeding on vertebrate hosts may be primarily responsible for POWV maintenance, and similarly could play a role in HRTV and BRBV circulation (Vogels et al. 2023). The vector competence studies discussed above support both cofeeding and transovarial transmission as possible mechanisms for HRTV and BRBV in A. americanum (Godsey et al. 2016, 2021).

Despite seroprevalence in domestic and wild animals, the status of HRTV and BRBV as potential veterinary pathogens remains unclear. Outside of certain mouse models, disease has not been observed in animals experimentally infected with HRTV. Disease in young sheep, goats, and cattle is observed with the related Bhanja bandavirus (Hubalek 1987, Hubalek et al. 2014). Severe epizootics in sheep, cattle, buffalo, and goats are associated with Rift Valley Fever virus, a phylogenetically related mosquito-borne phlebovirus (HRTV and DBV were formerly grouped with the phleboviruses) (Meegan et al. 1979, House et al. 1992, Madani et al. 2003, Chevalier et al. 2010, Hubalek et al. 2014, Nyakarahuka et al. 2023). Of the thogotoviruses, only the eponymous THOV has been shown to produce disease in animals, causing leukopenia in cattle and abortion and fever in sheep (Davies et al. 1984, Sang et al. 2006b). Further exploration of HRTV and BRBV in the context of wild animal host associations and assessment of disease potential in domestic, agricultural, and companion animals is warranted.

Vector Control and Surveillance

Forum articles previously published in the Journal of Medical Entomology, Special Collection: The Rise of Ticks and Tick-Borne Diseases were devoted to vector control and surveillance practices (Eisen and Paddock 2021, Eisen and Stafford 2021, Mader et al. 2021, Tokarz and Lipkin 2021). The same theories, practices, challenges, and conclusions presented in those manuscripts are directly applicable to the current status of HRTV and BRBV emergence. We will attempt to summarize some of the major points, more elegantly stated and discussed in detail in the original manuscripts, relative to HRTV and BRBV. Regarding vector control, broadcast acaricidal treatments may be effective at reducing the local tick population regardless of tick species; however, the scale of operation, the effect on nontarget organisms, and acaricidal resistance are important considerations (Eisen and Stafford 2021). Furthermore, the treatment does not necessarily reduce the risk of tick bite or disease beyond the application site (Hinckley et al. 2016). Results recently published from the Tick Project (Fischhoff et al. 2017, Keesing et al. 2022) illustrate these challenges. In a large-scale, placebo-controlled study in Dutchess County, NY, an area hyperendemic for I. scapularis and the pathogens they transmit, the use of fipronil-treated bait boxes and a fungal spray (Met52) was assessed for effectiveness at reducing I. scapularis abundance, tick encounters of humans and pets, and cases of tick-borne disease. Results indicated a significant reduction of questing ticks within treatment areas and a lower number of pet-tick encounters and incidence of tick-borne disease in pets, but no concomitant significant reduction in human-tick encounters or tick-borne disease in humans was achieved (Keesing et al. 2022). Obviously, this system is not completely analogous to the A. americanum/HRTV and BRBV transmission cycle, but merely demonstrates the complexities and limitations of tick management. In fact, there are fewer studies of acaricidal control of A. americanum compared to I. scapularis (Eisen and Stafford 2021), though the use of 4-poster pesticide applicators in certain areas and situations have been shown to manage A. americanum populations (Schulze et al. 2007, Pound et al. 2009, Williams et al. 2021) and reduced Ehrlichia spp. in one of the studies (Williams et al. 2021).

An accurate assessment of the risk of tick-borne disease is dependent on robust surveillance programs. It includes active and passive tick surveillance for the creation of species distribution maps and monitoring of tick populations, tick-borne pathogen testing, and reporting (Beard et al. 2021, Eisen and Paddock 2021). These practices are not universal or standardized and are certainly not available nationwide (Beard et al. 2021, Eisen and Paddock 2021, Mader et al. 2021). Further, surveillance programs rely on inconsistent funding streams and these programs do not account for human behavior. The CDC currently maintains a user-friendly dashboard of tick species and associated pathogen distribution that could be easily modified to include HRTV and BRBV (CDC 2023c, 2023d, 2023e).

Unfortunately, emerging tick-borne pathogens, like HRTV and BRBV, are often discovered as a result of serious or severe clinical infections. Now that HRTV and BRBV have been identified across the range (and beyond) of A. americanum, it is imperative that clinicians are aware of these pathogens and the signs and symptoms of disease. HRTV and BRBV should be included in tick and tick-borne pathogen surveillance programs that have the capacity. The utility of testing hunter-harvested white-tailed deer for the presence of vector-borne pathogens is well established (Emmons 1968, Issel et al. 1972, Piesman et al. 1979, Campbell et al. 1989, McLean et al. 1996, Farajollahi et al. 2004, Mutebi et al. 2011, Pedersen et al. 2017, Dupuis et al. 2020). This is especially true for tick-borne pathogens such as HRTV and BRBV with low tick infection rates and areas without tick surveillance (Riemersma and Komar 2015, Clarke et al. 2018b, Komar et al. 2020, Dupuis et al. 2021, 2023). A fundable national or multi-regional testing program facilitated or coordinated by state and federal partners, perhaps modeled on the current regional Centers of Excellence, would complement existing tick surveillance programs and citizen science initiatives such as tick blitz (Foley et al. 2023) and tick submission/testing at commercial laboratories. Leveraging federal and state natural resource divisions, local fish and game clubs, and high-volume venison processors would provide an ample supply of samples (blood and ticks) to define the distribution of HRTV and BRBV and could be easily modified for other endemic and emerging tick- and mosquito-borne pathogens.

Conclusion

HRTV and BRBV are emerging threats to public health evidenced by the potential to cause severe disease in humans and recent expansion into novel ticks and regions of North America. These viruses highlight the need for expanded viral surveillance, clinical testing, and assay development to further inform vector control and clinical diagnosis and treatment. Gaps also remain concerning HRTV and BRBV molecular mechanisms that contribute to tick transmission and host competence. Studies aimed at understanding disease pathogenesis in humans and wildlife are also needed to inform therapeutic design. Expanding the number of available HRTV and BRBV strains and molecular tools, as well as the availability of robust in vivo models, is necessary to address these knowledge gaps. Institutional frameworks to support these efforts have been demonstrated at the local, regional, and national level and require continued support to address HRTV and BRBV emergence and future emergent tick-borne viruses.

References