Serological Prevalence of Crimean-Congo Hemorrhagic Fever Virus Infection in Small Ruminants and Cattle in The Gambia.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a widely distributed tickborne zoonotic agent that infects a variety of host species. There is a lack of information on the true geographic distribution of the prevalence and risk of CCHFV in West Africa. A countrywide cross-sectional study involving 1413 extensively managed indigenous small ruminants and cattle at livestock sales markets and in village herds, respectively, was carried out in The Gambia. In sheep, an overall anti-CCHFV antibody prevalence of 18.9% (95% CI: 15.5-22.8%), goats 9.0% (95% CI: 6.7-11.7%), and cattle 59.9% (95% CI: 54.9-64.7%) was detected. Significant variation (p < 0.05) in the prevalence of anti-CCHFV antibodies at sites in the five administrative regions (sheep: 4.8-25.9%; goats: 1.8-17.1%) and three agroecological zones (sheep: 8.9-32.9%; goats: 4.1-18.0%) was also observed. Comparatively, higher anti-CCHFV antibody prevalence was detected in cattle (33.3-84.0%) compared to small ruminants (1.8-8.1%). This study represents the first countrywide investigation of the seroprevalence of CCHFV in The Gambia, and the results suggest potential circulation and endemicity of the virus in the country. These data provide critical information vital to the development of informed policies for the surveillance, diagnosis, and control of CCFHV infection in The Gambia and the region.

Evaluation of oral fluid as an aggregate sample for early detection of African swine fever virus using four independent pen-based experimental studies.

The sustained spread of African swine fever (ASF) virus throughout much of the world has made ASF a global animal health priority, with an increased emphasis on enhancing preparedness to prevent, detect and respond to a potential outbreak of ASF virus (ASFV). In the event of ASFV entry to the North American swine population, enhanced surveillance and diagnostic testing strategies will be critical to facilitate progressive response and eradication of the disease. Compared to individual animal sampling, pen-based oral fluid collection for active surveillance is a non-invasive alternative that is less resource and time-intensive. To evaluate the feasibility of using rope-based oral fluid for early detection of ASFV, four independent animal experiments were conducted in weaned pigs housed in numbers that mimic the industry settings, utilising either highly virulent ASFV Georgia 2007/1 strain or moderately virulent ASFV Malta'78 strain. Pen-based oral fluid and individual oropharyngeal swabs were collected daily and blood samples from each animal were collected every other day. All samples were subsequently tested for ASFV by real-time PCR. ASFV genome was detected in individual blood samples as early as one day post-infection and detected in oral fluids at low-to-moderate levels as early as 3-5 days post-infection in all four independent experiments. These results suggest that pen-based oral fluid samples may be used to supplement the use of traditional samples for rapid detection of ASFV during ASF surveillance.

Outbreak of rabbit hemorrhagic disease virus 2 in the southwestern United States: first detections in southern California.

An outbreak of rabbit hemorrhagic disease virus 2 (RHDV2)-associated disease occurred in the southwestern United States following its first detection in New Mexico in March 2020. The disease spread throughout several states and was diagnosed for the first time in California on May 11, 2020, in a black-tailed jackrabbit (Lepus californicus). The following day, the California Department of Food and Agriculture (CDFA) issued an order banning the entrance into California of several lagomorph species and their products from any state in which the disease had been detected in the last 12 mo. RHDV2 is a threat to wild lagomorph species in California, including the endangered riparian brush rabbit (Sylvilagus bachmani riparius). Therefore, the California Department of Fish and Wildlife (CDFW) started tracking any mortality event in wild lagomorph populations. As of August 9, 2020, RHDV2 had been detected in wild and domestic lagomorphs of several counties in southern California that were submitted to the California Animal Health and Food Safety laboratory system by the CDFA or the CDFW. These positive cases included 2 additional black-tailed jackrabbits and 3 desert cottontail rabbits (Sylvilagus audubonii). In addition, the infection spilled over to domestic populations, whereby it was confirmed on July 10, 2020, in a domestic rabbit (Oryctolagus cuniculus).

Validation of a binary ethylenimine (BEI) inactivation procedure for biosafety treatment of foot-and-mouth disease viruses (FMDV), vesicular stomatitis viruses (VSV), and swine vesicular disease virus (SVDV).

Binary ethylenimine (BEI) has been widely used as a virucide to inactivate viruses. For regulatory exclusion of a select agent, the United States Federal Select Agent Program (FSAP) requires an inactivation procedure that renders a select agent non-viable but allows the select agent to retain antigenic characteristics for future use must be validated, and the inactivated agent must be confirmed by a viability testing. In this curve-based validation study, we examined impacts of BEI concentration, treatment temperature, and time on our in-house inactivation procedures of Foot-and-Mouth Disease Virus (FMDV), Vesicular Stomatitis Virus (VSV), and Swine Vesicular Disease Virus (SVDV). The inactivation efficacy was confirmed by virus titration and 3 consecutive blind passages on the monolayers of susceptible cells. A linear correlation between the virus titer reduction and BEI concentration, treatment time, and temperature was established. The results confirmed our in-house BEI inactivation procedure of two doses of 1.5 mM BEI treatment at 37 °C, 1st dose for 24 h, then 2nd dose for 6 more hours for a total of 30 h BEI contact time, can ensure complete inactivation of FMDV, VSV, and SVDV.

Development of a real-time PCR assay for detection of African swine fever virus with an endogenous internal control.

Real-time PCR assays are highly sensitive, specific and rapid techniques for the identification of ASF virus (ASFV) (Section 3.8, OIE Terrestrial Manual, 2019). Although an ASFV p72 gene-based real-time PCR assay (a.k.a. the Zsak assay) (Journal of Clinical Microbiology, 2005, 43, 112) has been widely used for ASFV detection, several more ASFV whole genome sequences have become available in the 15 years since the design of the Zsak assay. In this study, we developed a new ASFV p72 gene-based real-time PCR after analysis of all currently available sequences of the p72 gene and multiplexed the new assay with a modified Zsak assay aiming to have a broader coverage of ASFV strain/isolates. To reduce false-negative detections, porcine house-keeping gene, beta actin (ACTB), was applied as an internal control. Eight ACTB sequences from the GenBank and 61 partial ACTB sequences generated in this study, and 1,012 p72 sequences from the GenBank and 23 p72 sequences generated at FADDL, were used for ACTB and ASFV primer and probe designs, respectively, to ensure broader host and ASFV coverage. Multiplexing ACTB in the reaction did not affect ASFV amplification. The multiplex assay was evaluated for strain/isolate coverage, sensitivity and specificity. The in silico analysis showed high ASFV strain/isolate coverage: 98.4% (978/994) of all p72 sequences currently available. The limit of detection (LOD) was 6 plasmid copies or 0.1-1 TCID50 /ml of ASFV isolates per reaction. Only targeted ASFV isolates and the viruses in the positive clinical samples were detected, indicating that the assay is highly specific (100% specificity). The test results of 26 ASFV isolates with different country origins showed that this newly developed multiplex assay performed better than the Zsak assay that has been widely accepted and used worldwide, indicating that it may be used as an alternative assay for ASFV detection.

Antigenic regions of African swine fever virus phosphoprotein P30.

African swine fever (ASF) is one of the most complex and lethally haemorrhagic viral diseases of swine, affecting all breeds and ages of pigs. In the absence of ASF vaccines, reliable laboratory diagnosis and restricted biosecurity are critical for disease prevention and control. A detection of ASF-specific antibodies in an unvaccinated pig is a good marker for the diagnosis of ASF. The immunoperoxidase test (IPT) is a sensitive test for detecting ASF virus (ASFV) antibodies. However, due to the complexity of the procedure, the IPT is only suitable to be used as a confirmatory test. The ASFV p30 protein-based enzyme-linked immunosorbent assay (ELISA) is widely used for ASFV antibody screening, but the sensitivity is not comparable to the IPT. It is essential to have a better understanding of the antigenic properties of ASFV p30 to improve p30-based serologic tests. In this study, we developed a panel of 21 monoclonal antibodies (mAbs) against ASFV p30. With 14 out of the 21 mAbs, we defined 4 antigenic regions that contain at least 4 linear epitopes. Nine of the 14 mAbs mapped to antigenic regions 3 and 4 reacted with p30 in all serologic methods tested in this study, such as indirect immunofluorescence assay (IFA), ELISA and Western blot. The antigenic regions 3 and 4 are highly conserved and immunodominant in host antibody response. These mAbs and the defined p30 antigenic regions 3 and 4 provide valuable tools for the development and improvement of ASF serologic assays.

Rapid Sequence-Based Characterization of African Swine Fever Virus by Use of the Oxford Nanopore MinION Sequence Sensing Device and a Companion Analysis Software Tool.

African swine fever virus (ASFV) is the causative agent of a severe and highly contagious viral disease of pigs that poses serious economic consequences to the swine industry due to the high mortality rate and impact on international trade. There is no effective vaccine to control African swine fever (ASF), and therefore, efficient disease control is dependent on early detection and diagnosis of ASFV. The large size of the ASFV genome (∼180 kb) has historically hindered efforts to rapidly obtain a full-genome sequence. Rapid acquisition of data is critical for characterization of the isolate and to support epidemiological efforts. Here, we investigated the capacity of the Oxford Nanopore MinION sequence sensing device to act as a rapid sequencing tool. When coupled with our novel companion software script, the African swine fever fast analysis sequencing tool (ASF-FAST), the analysis of output data was performed in real time. Complete ASFV genome sequences were generated from cell culture isolates and blood samples obtained from experimentally infected pigs. Removal of the host-methylated DNA from the extracted nucleic acid facilitated rapid ASFV sequence identification, with reads specific to ASFV detected within 6 min after the initiation of sequencing. Regardless of the starting material, sufficient sequence was available for complete genome resolution (up to 100%) within 10 min. Overall, this paper highlights the use of Nanopore sequencing technology in combination with the ASF-FAST software for the purpose of rapid and real-time resolution of the full ASFV genome from a diagnostic sample.

Ebola Virus Disease Diagnostics, Sierra Leone: Analysis of Real-time Reverse Transcription-Polymerase Chain Reaction Values for Clinical Blood and Oral Swab Specimens.

During the Ebola virus outbreak of 2013-2016, the Viral Special Pathogens Branch field laboratory in Sierra Leone tested approximately 26 000 specimens between August 2014 and October 2015. Analysis of the B2M endogenous control Ct values showed its utility in monitoring specimen quality, comparing results with different specimen types, and interpretation of results. For live patients, blood is the most sensitive specimen type and oral swabs have little diagnostic utility. However, swabs are highly sensitive for diagnostic testing of corpses.

Effect of Vandetanib on Andes virus survival in the hamster model of Hantavirus pulmonary syndrome.

Hantavirus pulmonary syndrome (HPS) is a severe disease caused by hantavirus infection of pulmonary microvascular endothelial cells leading to microvascular leakage, pulmonary edema, pleural effusion and high case fatality. Previously, we demonstrated that Andes virus (ANDV) infection caused up-regulation of vascular endothelial growth factor (VEGF) and concomitant downregulation of the cellular adhesion molecule VE-cadherin leading to increased permeability. Analyses of human HPS-patient sera have further demonstrated increased circulating levels of VEGF. Here we investigate the impact of a small molecule antagonist of the VEGF receptor 2 (VEGFR-2) activation in vitro, and overall impact on survival in the Syrian hamster model of HPS.

Humanized Mouse Model of Ebola Virus Disease Mimics the Immune Responses in Human Disease.

Animal models recapitulating human Ebola virus disease (EVD) are critical for insights into virus pathogenesis. Ebola virus (EBOV) isolates derived directly from human specimens do not, without adaptation, cause disease in immunocompetent adult rodents. Here, we describe EVD in mice engrafted with human immune cells (hu-BLT). hu-BLT mice developed EVD following wild-type EBOV infection. Infection with high-dose EBOV resulted in rapid, lethal EVD with high viral loads, alterations in key human antiviral immune cytokines and chemokines, and severe histopathologic findings similar to those shown in the limited human postmortem data available. A dose- and donor-dependent clinical course was observed in hu-BLT mice infected with lower doses of either Mayinga (1976) or Makona (2014) isolates derived from human EBOV cases. Engraftment of the human cellular immune system appeared to be essential for the observed virulence, as nonengrafted mice did not support productive EBOV replication or develop lethal disease. hu-BLT mice offer a unique model for investigating the human immune response in EVD and an alternative animal model for EVD pathogenesis studies and therapeutic screening.

Ebola Virus Diagnostics: The US Centers for Disease Control and Prevention Laboratory in Sierra Leone, August 2014 to March 2015.

In August 2014, the Viral Special Pathogens Branch of the US Centers for Disease Control and Prevention established a field laboratory in Sierra Leone in response to the ongoing Ebola virus outbreak. Through March 2015, this laboratory tested >12 000 specimens from throughout Sierra Leone. We describe the organization and procedures of the laboratory located in Bo, Sierra Leone.

An assisted living facility curriculum to introduce geriatrics to first-year medical students.

Many U.S. medical schools have developed curricula in geriatric medicine to address the growing older adult population. At our university, the authors have integrated an assisted living facility (ALF) program into a required first-year clinical skills course. During the 2011 to 2012 academic year, an electronic survey was distributed to 109 first-year medical students prior to and after the program. Eighty-eight percent and 85% of students completed the pre- and postintervention survey, respectively. Students reported a positive attitude toward caring for older adults (92.5% post- vs. 80.2% preintervention), an understanding of the medical and social needs of older adults (89.2% post- vs. 38.5% preintervention), an acquisition of the skills to assess the health of older adults (71% post- vs. 14.5% preintervention), and an understanding of ALFs as nonmedical supportive housing (92.5% post- vs. 70.8% preintervention). The authors' curriculum offers an innovative method to integrate geriatrics education early in medical education and to involve medical students in their community.

Kyasanur Forest disease virus infection in mice is associated with higher morbidity and mortality than infection with the closely related Alkhurma hemorrhagic fever virus.

BACKGROUND: Kyasanur Forest disease virus (KFDV) and Alkhurma hemorrhagic fever virus (AHFV) are closely related members of the Flavivirus genus and are important causes of human disease in India and the Arabian Peninsula, respectively. Despite high genetic similarity, the viruses have distinctly different host ranges and ecologies. Human cases of KFDV or AHFV develop a spectrum of disease syndromes ranging from liver pathology to neurologic disease. Case reports suggest KFDV is more commonly associated with hepatic and gastrointestinal manifestations whereas AHFV is more commonly associated with neurologic disease. METHODOLOGY/PRINCIPAL FINDINGS: Inoculation of three immunocompetent laboratory mouse strains revealed that KFDV was consistently more lethal than AHFV. In subsequent studies utilizing C57BL/6J mice, we demonstrated that KFDV infection was associated with higher viral loads and significantly higher mortality. KFDV-infected mice rapidly developed more severe disease than AHFV-infected mice, as evidenced by significant abnormalities on clinical chemistry panels and more severe pathology in the brain and gastrointestinal tract. CONCLUSIONS/SIGNIFICANCE: Infections of C57BL/6J mice with KFDV or AHFV resulted in clinical disease syndromes that closely approximate the diseases seen in human cases. Despite high genetic similarity, there were clear differences in survival, viral kinetics, clinical chemistry data and histology. These results suggest that distinct mouse models for AHFV and KFDV are necessary in order to gain a better understanding of the unique pathogenesis of each virus, as well as to provide platforms for testing promising vaccines and therapeutics.

Rift valley Fever virus encephalitis is associated with an ineffective systemic immune response and activated T cell infiltration into the CNS in an immunocompetent mouse model.

BACKGROUND: Rift Valley fever virus (RVFV) causes outbreaks of severe disease in livestock and humans throughout Africa and the Arabian Peninsula. In people, RVFV generally causes a self-limiting febrile illness but in a subset of individuals, it progresses to more serious disease. One manifestation is a delayed-onset encephalitis that can be fatal or leave the afflicted with long-term neurologic sequelae. In order to design targeted interventions, the basic pathogenesis of RVFV encephalitis must be better understood. METHODOLOGY/PRINCIPAL FINDINGS: To characterize the host immune responses and viral kinetics associated with fatal and nonfatal infections, mice were infected with an attenuated RVFV lacking NSs (ΔNSs) that causes lethal disease only when administered intranasally (IN). Following IN infection, C57BL/6 mice developed severe neurologic disease and succumbed 7-9 days post-infection. In contrast, inoculation of ΔNSs virus subcutaneously in the footpad (FP) resulted in a subclinical infection characterized by a robust immune response with rapid antibody production and strong T cell responses. IN-inoculated mice had delayed antibody responses and failed to clear virus from the periphery. Severe neurological signs and obtundation characterized end stage-disease in IN-inoculated mice, and within the CNS, the development of peak virus RNA loads coincided with strong proinflammatory responses and infiltration of activated T cells. Interestingly, depletion of T cells did not significantly alter survival, suggesting that neurologic disease is not a by-product of an aberrant immune response. CONCLUSIONS/SIGNIFICANCE: Comparison of fatal (IN-inoculated) and nonfatal (FP-inoculated) ΔNSs RVFV infections in the mouse model highlighted the role of the host immune response in controlling viral replication and therefore determining clinical outcome. There was no evidence to suggest that neurologic disease is immune-mediated in RVFV infection. These results provide important insights for the future design of vaccines and therapeutic options.

Inhibitors of the tick-borne, hemorrhagic fever-associated flaviviruses.

No antiviral therapies are available for the tick-borne flaviviruses associated with hemorrhagic fevers: Kyasanur Forest disease virus (KFDV), both classical and the Alkhurma hemorrhagic fever virus (AHFV) subtype, and Omsk hemorrhagic fever virus (OHFV). We tested compounds reported to have antiviral activity against members of the Flaviviridae family for their ability to inhibit AHFV replication. 6-Azauridine (6-azaU), 2'-C-methylcytidine (2'-CMC), and interferon alpha 2a (IFN-α2a) inhibited the replication of AHFV and also KFDV, OHFV, and Powassan virus. The combination of IFN-α2a and 2'-CMC exerted an additive antiviral effect on AHFV, and the combination of IFN-α2a and 6-azaU was moderately synergistic. The combination of 2'-CMC and 6-azaU was complex, being strongly synergistic but with a moderate level of antagonism. The antiviral activity of 6-azaU was reduced by the addition of cytidine but not guanosine, suggesting that it acted by inhibiting pyrimidine biosynthesis. To investigate the mechanism of action of 2'-CMC, AHFV variants with reduced susceptibility to 2'-CMC were selected. We used a replicon system to assess the substitutions present in the selected AHFV population. A double NS5 mutant, S603T/C666S, and a triple mutant, S603T/C666S/M644V, were more resistant to 2'-CMC than the wild-type replicon. The S603T/C666S mutant had a reduced level of replication which was increased when M644V was also present, although the replication of this triple mutant was still below that of the wild type. The S603 and C666 residues were predicted to lie in the active site of the AHFV NS5 polymerase, implicating the catalytic center of the enzyme as the binding site for 2'-CMC.

Rift Valley fever virus clearance and protection from neurologic disease are dependent on CD4+ T cell and virus-specific antibody responses.

Rift Valley fever virus (RVFV) causes outbreaks of severe disease in people and livestock throughout Africa and the Arabian Peninsula. Human RVFV infections generally manifest as a self-limiting febrile illness, but in some individuals, the disease can progress to a fatal encephalitis or hemorrhagic syndrome. Little is known about the host characteristics that predispose development of more severe disease. Early in infection, interferon-mediated antiviral responses are critical for controlling RVFV replication, but the roles of downstream adaptive immune responses in determining clinical outcome have not been examined. Here, using a C57BL/6 mouse disease model, we evaluated the roles of B cells and T cells in RVFV pathogenesis. Given the profound inhibition of the innate response by the viral NSs protein and rapid course of wild-type infection, we utilized an attenuated RVFV lacking NSs to examine host responses following primary infection. Experiments utilizing B-cell-deficient mice or targeted T cell depletions of wild-type mice demonstrated that B cells and CD4(+) T cells, but not CD8(+) T cells, were critical for mediating viral clearance, even in the presence of a functional innate response. One-third of CD4-depleted mice developed severe neurologic disease following infection, in contrast to virus-infected mock-depleted mice that showed no clinical signs. CD4(+) T cells were required for robust IgG and neutralizing antibody responses that correlated with RVFV clearance from peripheral tissues. Furthermore, CD4-depleted mice demonstrated significantly stronger proinflammatory responses relative to controls, suggesting CD4(+) T cells regulate immune responses to RVFV infection. Together, these results indicate CD4(+) T cells are critical determinants of RVFV pathogenesis and play an important role in preventing onset of neurologic disease.

Severe hemorrhagic fever in strain 13/N guinea pigs infected with Lujo virus.

Lujo virus (LUJV) is a novel member of the Arenaviridae family that was first identified in 2008 after an outbreak of severe hemorrhagic fever (HF). In what was a small but rapidly progressing outbreak, this previously unknown virus was transmitted from the critically ill index patient to 4 attending healthcare workers. Four persons died during this outbreak, for a total case fatality of 80% (4/5). The suspected rodent source of the initial exposure to LUJV remains a mystery. Because of the ease of transmission, high case fatality, and novel nature of LUJV, we sought to establish an animal model of LUJV HF. Initial attempts in mice failed, but infection of inbred strain 13/N guinea pigs resulted in lethal disease. A total of 41 adult strain 13/N guinea pigs were infected with either wild-type LUJV or a full-length recombinant LUJV. Results demonstrated that strain 13/N guinea pigs provide an excellent model of severe and lethal LUJV HF that closely resembles what is known of the human disease. All infected animals experienced consistent weight loss (3-5% per day) and clinical illness characterized by ocular discharge, ruffled fur, hunched posture, and lethargy. Uniform lethality occurred by 11-16 days post-infection. All animals developed disseminated LUJV infection in various organs (liver, spleen, lung, and kidney), and leukopenia, lymphopenia, thrombocytopenia, coagulopathy, and elevated transaminase levels. Serial euthanasia studies revealed a temporal pattern of virus dissemination and increasing severity of disease, primarily targeting the liver, spleen, lungs, and lower gastrointestinal tract. Establishing an animal LUJV model is an important first step towards understanding the high pathogenicity of LUJV and developing vaccines and antiviral therapeutic drugs for this highly transmissible and lethal emerging pathogen.

Single-dose immunization with virus replicon particles confers rapid robust protection against Rift Valley fever virus challenge.

Rift Valley fever virus (RVFV) causes outbreaks of severe disease in people and livestock throughout Africa and the Arabian Peninsula. The potential for RVFV introduction outside the area of endemicity highlights the need for fast-acting, safe, and efficacious vaccines. Here, we demonstrate a robust system for the reverse genetics generation of a RVF virus replicon particle (VRP(RVF)) vaccine candidate. Using a mouse model, we show that VRP(RVF) immunization provides the optimal balance of safety and single-dose robust efficacy. VRP(RVF) can actively synthesize viral RNA and proteins but lacks structural glycoprotein genes, preventing spread within immunized individuals and reducing the risk of vaccine-induced pathogenicity. VRP(RVF) proved to be completely safe following intracranial inoculation of suckling mice, a stringent test of vaccine safety. Single-dose subcutaneous immunization with VRP(RVF), although it is highly attenuated, completely protected mice against a virulent RVFV challenge dose which was 100,000-fold greater than the 50% lethal dose (LD(50)). Robust protection from lethal challenge was observed by 24 h postvaccination, with 100% protection induced in as little as 96 h. We show that a single subcutaneous VRP(RVF) immunization initiated a systemic antiviral state followed by an enhanced adaptive response. These data contrast sharply with the much-reduced survivability and immune responses observed among animals immunized with nonreplicating viral particles, indicating that replication, even if confined to the initially infected cells, contributes substantially to protective efficacy at early and late time points postimmunization. These data demonstrate that replicon vaccines successfully bridge the gap between safety and efficacy and provide insights into the kinetics of antiviral protection from RVFV infection.

Ancient ancestry of KFDV and AHFV revealed by complete genome analyses of viruses isolated from ticks and mammalian hosts.

BACKGROUND: Alkhurma hemorrhagic fever virus (AHFV) and Kyasanur forest disease virus (KFDV) cause significant human disease and mortality in Saudi Arabia and India, respectively. Despite their distinct geographic ranges, AHFV and KFDV share a remarkably high sequence identity. Given its emergence decades after KFDV, AHFV has since been considered a variant of KFDV and thought to have arisen from an introduction of KFDV to Saudi Arabia from India. To gain a better understanding of the evolutionary history of AHFV and KFDV, we analyzed the full length genomes of 16 AHFV and 3 KFDV isolates. METHODOLOGY/PRINCIPAL FINDINGS: Viral genomes were sequenced and compared to two AHFV sequences available in GenBank. Sequence analyses revealed higher genetic diversity within AHFVs isolated from ticks than human AHFV isolates. A Bayesian coalescent phylogenetic analysis demonstrated an ancient divergence of AHFV and KFDV of approximately 700 years ago. CONCLUSIONS/SIGNIFICANCE: The high sequence diversity within tick populations and the presence of competent tick vectors in the surrounding regions, coupled with the recent identification of AHFV in Egypt, indicate possible viral range expansion or a larger geographic range than previously thought. The divergence of AHFV from KFDV nearly 700 years ago suggests other AHFV/KFDV-like viruses might exist in the regions between Saudi Arabia and India. Given the human morbidity and mortality associated with these viruses, these results emphasize the importance of more focused study of these significant public health threats.