Pathogenesis and virulence of Heartland virus.

Heartland virus (HRTV), an emerging tick-borne pathogenic bunyavirus, has been a concern since 2012, with an increasing incidence, expanding geographical distribution, and high pathogenicity in the United States. Infection from HRTV results in fever, thrombocytopenia, and leucopenia in humans, and in some cases, symptoms can progress to severe outcomes, including haemorrhagic disease, multi-organ failure, and even death. Currently, no vaccines or antiviral drugs are available for treatment of the HRTV disease. Moreover, little is known about HRTV-host interactions, viral replication mechanisms, pathogenesis and virulence, further hampering the development of vaccines and antiviral interventions. Here, we aimed to provide a brief review of HRTV epidemiology, molecular biology, pathogenesis and virulence on the basis of published article data to better understand this virus and provide clues for further study.

Widespread Circulation of Tick-Borne Viruses in Virginia-Evidence of Exposure to Heartland, Bourbon, and Powassan Viruses in Wildlife and Livestock.

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.

Modeling Heartland virus disease in mice and therapeutic intervention with 4'-fluorouridine.

Heartland virus (HRTV) is an emerging tick-borne bandavirus that causes a febrile illness of varying severity in humans, with cases reported in eastern and midwestern regions of the United States. No vaccines or approved therapies are available to prevent or treat HRTV disease. Here, we describe the genetic changes, natural history of disease, and pathogenesis of a mouse-adapted HRTV (MA-HRTV) that is uniformly lethal in 7- to 8-week-old AG129 mice at low challenge doses. We used this model to assess the efficacy of the ribonucleoside analog, 4'-fluorouridine (EIDD-2749), and showed that once-daily oral treatment with 3 mg/kg of drug, initiated after the onset of disease, protects mice against lethal MA-HRTV challenge and reduces viral loads in blood and tissues. Our findings provide insights into HRTV virulence and pathogenesis and support further development of EIDD-2749 as a therapeutic intervention for HRTV disease. IMPORTANCE: More than 60 cases of HRTV disease spanning 14 states have been reported to the United States Centers for Disease Control and Prevention. The expanding range of the Lone Star tick that transmits HRTV, the growing population of at-risk persons living in geographic areas where the tick is abundant, and the lack of antiviral treatments or vaccines raise significant public health concerns. Here, we report the development of a new small-animal model of lethal HRTV disease to gain insight into HRTV pathogenesis and the application of this model for the preclinical development of a promising new antiviral drug candidate, EIDD-2749. Our findings shed light on how the virus causes disease and support the continued development of EIDD-2749 as a therapeutic for severe cases of HRTV infection.

Heartland Virus Disease-An Underreported Emerging Infection.

First recognized 15 years ago, Heartland virus disease (Heartland) is a tickborne infection contracted from the transmission of Heartland virus (HRTV) through tick bites from the lone star tick (Amblyomma americanum) and potentially other tick species. Heartland symptoms include a fever <100.4 °F, lethargy, fatigue, headaches, myalgia, a loss of appetite, nausea, diarrhea, weight loss, arthralgia, leukopenia and thrombocytopenia. We reviewed the existing peer-reviewed literature for HRTV and Heartland to more completely characterize this rarely reported, recently discovered illness. The absence of ongoing serosurveys and targeted clinical and tickborne virus investigations specific to HRTV presence and Heartland likely contributes to infection underestimation. While HRTV transmission occurs in southern and midwestern states, the true range of this infection is likely larger than now understood. The disease's proliferation benefits from an expanded tick range due to rising climate temperatures favoring habitat expansion. We recommend HRTV disease be considered in the differential diagnosis for patients with a reported exposure to ticks in areas where HRTV has been previously identified. HRTV testing should be considered early for those matching the Heartland disease profile and nonresponsive to initial broad-spectrum antimicrobial treatment. Despite aggressive supportive therapy, patients deteriorating to sepsis early in the course of the disease have a very grim prognosis.

Computer-Selected Antiviral Compounds: Assessing In Vitro Efficacies against Rift Valley Fever Virus.

Rift Valley fever is a zoonotic viral disease transmitted by mosquitoes, impacting both humans and livestock. Currently, there are no approved vaccines or antiviral treatments for humans. This study aimed to evaluate the in vitro efficacy of chemical compounds targeting the Gc fusion mechanism. These compounds were identified through virtual screening of millions of commercially available small molecules using a structure-based artificial intelligence bioactivity predictor. In our experiments, a pretreatment with small molecule compounds revealed that 3 out of 94 selected compounds effectively inhibited the replication of the Rift Valley fever virus MP-12 strain in Vero cells. As anticipated, these compounds did not impede viral RNA replication when administered three hours after infection. However, significant inhibition of viral RNA replication occurred upon viral entry when cells were pretreated with these small molecules. Furthermore, these compounds exhibited significant inhibition against Arumowot virus, another phlebovirus, while showing no antiviral effects on tick-borne bandaviruses. Our study validates AI-based virtual high throughput screening as a rational approach for identifying effective antiviral candidates for Rift Valley fever virus and other bunyaviruses.

Pathological and virological findings of type I interferon receptor knockout mice upon experimental infection with Heartland virus.

Heartland virus (HRTV) causes generalized symptoms, severe shock, and multiple organ failure. We previously reported that interferon-α/ß receptor knockout (IFNAR-/-) mice infected intraperitoneally with 1 × 107 tissue culture-infective dose (TCID50) of HRTV died, while those subcutaneously infected with the same dose of HRTV did not. The pathophysiology of IFNAR-/- mice infected with HRTV and the mechanism underlying the difference in disease severity, which depends on HRTV infection route, were analyzed in this study. The liver, spleen, mesenteric and axillary lymph nodes, and gastrointestinal tract of intraperitoneally (I.P.) infected mice had pathological changes; however, subcutaneously (S.C.) infected mice only had pathological changes in the axillary lymph node and gastrointestinal tract. HRTV RNA levels in the mesenteric lymph node, lung, liver, spleen, kidney, stomach, intestine, and blood were significantly higher in I.P. infected mice than those in S.C. infected mice. Chemokine ligand-1 (CXCL-1), tumor necrosis factor (TNF)-α, interleukin (IL)-12, interferon (IFN)-γ, and IL-10 levels in plasma of I.P. infected mice were higher than those of S.C. infected mice. These results indicated that high levels of viral RNA and the induction of inflammatory responses in HRTV-infected IFNAR-/- mice may be associated with disease severity.

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

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.

Fatal Case of Heartland Virus Disease Acquired in the Mid-Atlantic Region, United States.

Heartland virus (HRTV) disease is an emerging tickborne illness in the midwestern and southern United States. We describe a reported fatal case of HRTV infection in the Maryland and Virginia region, states not widely recognized to have human HRTV disease cases. The range of HRTV could be expanding in the United States.

Prevalence of Bourbon and Heartland viruses in field collected ticks at an environmental field station in St. Louis County, Missouri, USA.

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.

Generation of Multiple Arbovirus-like Particles Using a Rapid Recombinant Vaccinia Virus Expression Platform.

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.

Characterization of proteome wide antigenic epitopes to design proteins specific and proteome-wide ensemble vaccines against heartland virus using structural vaccinology and immune simulation approaches.

Heartland virus is a single-stranded negative-sense RNA virus that infects humans and causes lethargy, myalgia, headaches, nausea, diarrhea, weight loss, arthralgia, loss of appetite, leukopenia, and easy bruising due thrombocytopenia. The unavailability of antiviral drugs for HRTV infection is a major obstacle to treat this infection, therefore supportive care management is adopted in the case of a severe ailment. In this scientific study, proteins specific and proteome-wide Helper T-cell (HTL), linear B cell, and cytotoxic T-cell (CTL) epitopes mapping joined together with suitable linkers to design multi-epitopes subunit vaccine (MEVC). The constructed four vaccines from nucleocapsid protein, replicase, glycoprotein and finally whole proteome-wide constructs demonstrated stronger antigenic and non-allergenic behavior. Physiochemical properties evaluation also reported easy and efficient expression and downstream analysis of the constructs. Molecular docking of these constructs with toll-like receptor 7 (TLR7) revealed good binding and further validation based on MM/GBSA also demonstrated stronger interaction between the vaccine constructs and TLR7. Moreover, in silico cloning reported CAI value of 0.96 for each construct and excellent GC contents percentage required for experimental analysis. Furthermore, immune simulation-based immune response surveillance revealed that upon the injection of antigen the primary and secondary antibodies were produced between 5 and 15 days, and a more robust neutralization of the antigen by the proteome-wide vaccine construct was observed. This research could pave the way for the development of dynamic and efficient vaccines that contain a unique mix of numerous HRTV derived antigenic peptides to control HRTV infection.

Isolation of Heartland Virus from Lone Star Ticks, Georgia, USA, 2019.

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.

Transovarial Transmission of Heartland Virus by Invasive Asian Longhorned Ticks under Laboratory Conditions.

We demonstrated experimental acquisition and transmission of Heartland bandavirus by Haemaphysalis longicornis ticks. Virus was detected in tick salivary gland and midgut tissues. A total of 80% of mice exposed to 1 infected tick seroconverted, suggesting horizontal transmission. H. longicornis ticks can transmit the virus in the transovarial mode.

Heartland Virus Transmission, Suffolk County, New York, USA.

During 2018, Heartland virus RNA was detected in an Amblyomma americanum tick removed from a resident of Suffolk County, New York, USA. The person showed seroconversion. Tick surveillance and white-tailed deer (Odocoileus virginianus) serosurveys showed widespread distribution in Suffolk County, emphasizing a need for disease surveillance anywhere A. americanum ticks are established or emerging.

A panel of real-time PCR assays for the detection of Bourbon virus, Heartland virus, West Nile virus, and Trypanosoma cruzi in major disease-transmitting vectors.

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.

Characterization of pseudotyped vesicular stomatitis virus bearing the heartland virus envelope glycoprotein.

The heartland virus (HRTV) is a novel phlebovirus that causes severe infections in the USA and closely related to the severe fever thrombocytopenia syndrome virus (SFTSV), a causative agent for SFTS in Asia. The entry mechanisms of HRTV remain unclear. Here, we developed the pseudotyped vesicular stomatitis virus bearing the HRTV glycoprotein (GP) (HRTVpv), and the antigenicity and the entry mechanisms of HRTV were analyzed. HRTVpv was neutralized by anti-SFTSV Gc antibody, but not the anti-SFTSV Gn antibodies. Entry of HRTVpv to cells was inhibited by bafilomycin A1 and dynasore, and but it was enhanced in cells overexpressed with C-type lectins. Production of infectious HRTVpv and SFTSVpv was reduced by Nn-DNJ, α-glucosidase inhibitor. The entry of HRTV occurs via pH- and dynamin-dependent endocytosis. Furthermore, Nn-DNJ may be a possible therapeutic agent against HRTV and SFTSV.

Isothermal Recombinase Polymerase Amplification-Lateral Flow Point-of-Care Diagnostic Test for Heartland Virus.

The detection of novel or re-emergent pathogens necessitates the development of rapid, easy-to-use diagnostic tests that can be readily adapted and utilized in both clinical laboratories and field settings. Heartland virus (HRTV) is the first pathogenic Phlebovirus responsible for serious and fatal cases in the United States. We developed a qualitative test based on recombinase-polymerase-amplification coupled with lateral flow reading (RPA-LF) for rapid detection of HRTV. The RPA-LF detected HRTV with a limit of detection of 1.19-1.54 plaque-forming unit equivalents/reaction. In addition, the RPA-LF was able to detect 0.6075 copies/µL of HRTV nucleoprotein gene-containing plasmid. We evaluated six clinical samples that were previously found to be real-time PCR positive for HRTV and found five out of six samples to be positive by RPA-LF, yielding 83.3% concordance with real-time PCR. All six samples had Ct values between 29 and 39 by real-time PCR. We also determined that the HRTV primers and probe do not cross-react with other tick-transmitted viruses such as Bourbon and Powassan, or other related viruses, including Lonestar tick virus and Sunday canyon virus (100% specificity). This is the first isothermal amplification test developed for a tick-borne virus, which will allow for rapid differentiation between HRTV and other pathogens producing similar clinical manifestations.

Development of diagnostic microsphere-based immunoassays for Heartland virus.

BACKGROUND: Heartland virus (HRTV), a recently reclassified member of the genus Bandavirus, family Phenuiviridae, was first isolated in 2009 from a Missouri farmer exhibiting leukopenia and thrombocytopenia with suspected ehrlichiosis. Since then, more HRTV cases have been diagnosed, and firstline laboratory diagnostic assays are needed to identify future infections Objectives. We sought to develop rapid and reliable IgM and IgG microsphere immunoassays (MIAs) to test sera of patients suspected of having HRTV infection, and to distinguish between recent and past infections. STUDY DESIGN: Heartland virus antigen was captured by an anti-HRTV monoclonal antibody covalently bound to microspheres. Antibodies in human sera from confirmed HRTV-positive and negative cases were reacted with the microsphere complexes and detected using a BioPlex® 200 instrument. Assay cutoffs were determined by receiver operator characteristic analysis of the normalized test output values, equivocal zones for each assay were defined, and sensitivities, specificities, accuracies, and imprecision values were calculated. RESULTS: Sensitivities, specificities and accuracies of the IgM and IgG MIAs were all >95 %. Both tests were precise within and between assay plates, and cross-reactivity with other arboviruses was not observed. CONCLUSIONS: HRTV IgM and IgG MIAs are accurate and rapid first-line methods to serologically identify recent and past HRTV infections.

The NF-κB inhibitor, SC75741, is a novel antiviral against emerging tick-borne bandaviruses.

Severe fever with thrombocytopenia syndrome virus (SFTSV) and Heartland virus (HRTV) cause viral hemorrhagic fever-like illnesses in humans due to an aberrant host inflammatory response, which contributes to pathogenesis. Here, we established two separate minigenome (MG) systems based on the M-segment of SFTSV and HRTV. Following characterization of both systems for SFTSV and HRTV, we used them as a platform to screen potential compounds that inhibit viral RNA synthesis. We demonstrated that the NF-κB inhibitor, SC75741, reduces viral RNA synthesis of SFTSV and HRTV using our MG platform and validated these results using infectious SFTSV and HRTV. These results may lead to the use of MG systems as potential screening systems for the identification of antiviral compounds and yield novel insights into host-factors that could play role in bandavirus transcription and replication.

Heartland Virus in Lone Star Ticks, Alabama, USA.

We detected Heartland virus (HRTV) in lone star nymphs collected in 2018 in northern Alabama, USA. Real-time reverse transcription PCR selective for the small segment of the HRTV genome and confirmatory sequencing of positive samples showed high identity with HRTV strains sequenced from Tennessee and Missouri.