Location: Zoonotic and Emerging Disease Research
2025 Annual Report
Objectives
Objective 1. Develop CCHF virus diagnostic tests for the early detection and surveillance of Crimean-Congo Hemorrhagic Fever Virus.
Develop viral detection methods for ticks.
Develop direct viral detection methods for cattle, sheep, and goats.
Develop antibody detection methods to determine CCHF exposure in cattle, sheep, goats, and relevant wildlife.
Objective 2. Determine mechanisms of CCHF transmission.
Develop CCHF tick and animal infection methods.
Develop CCHF tick-animal transmission models.
Objective 3. Investigate the epidemiology of CCHF and the role of ticks in maintaining reservoirs of infection in endemic settings.
Determine the prevalence of CCHF in vector and animal hosts in endemic areas.
Determine the competence of hard tick species in endemic areas.
Determining the risk for the establishment of tick host vector in the United States considering climatic and ecological conditions.
Approach
The research addresses the following research components in the 2022-2027 Animal Health National Program (NP 103) Action Plan: 1) Component 1: Biodefense, Problem Statement 1A, Control and eradicate foreign animal diseases. The research addresses ARS Strategic Plan Goal 4.3 and the following Performance Measure: Provide scientific information to protect animals, humans, and property from the negative effects of pests and infectious diseases, and develop and transfer tools to the agricultural community, commercial partners, and government agencies to control or eradicate domestic and exotic diseases and pests that affect animal and human health.
Progress Report
FY25 marked a transformative year for the Crimean-Congo Hemorrhagic Fever virus (CCHFV) research program, with robust progress across surveillance, diagnostics, and countermeasure development. Fieldwork in Africa, Europe, and the U.S. generated large-scale tick and livestock datasets that will inform future risk models. Diagnostic innovations brought new tools closer to field deployment, including assays that function without cold chains or laboratory infrastructure. Foundational immunology and vaccine research advanced rapidly and progress was made defining the ecological and molecular drivers of transmission. The performed work strengthens USDAs efforts to protect U.S. agriculture fortifies biosecurity and preparedness stances.
Objective 1: Develop CCHF virus diagnostic tests for the early detection and surveillance of CCHFV.
To improve diagnostic access, projects are focusing on portable, sensitive, and easy-to-use diagnostic tools. A major milestone was nearing completion of a plug-and-play software platform for variant detection from sequencing data. Designed for use by reference labs, this tool supports rapid identification of both known and novel CCHFV strains.
Field-deployable assays: loop-mediated isothermal amplification (LAMP) and clustered regularly interspaced short palindromic repeats (CRISPR)-based systems are being optimized for stability and transport. Buffer formulations were created to minimize clotting and inactivate pathogens at the point of collection, enabling pen-side diagnostics in resource-limited settings and enhancing safety.
Spectral interference techniques are being developed to distinguish between infected and uninfected ticks based on chemical signatures. This method may serve as a non-molecular triage tool to prioritize samples for further testing.
Peptide-based serological assays were advanced for the differentiation of CCHFV. Short-peptide phage display platforms were also developed for pan-Nairoviruses, and other pathogens with similar presentation or geographic distribution, enabling multiplexed evaluation of immune responses in animals and humans.
Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (Maldi-TOF) platforms were upgraded and validated for tick identification and are being adapted to detect viral infection of vectors.
Objective 2. Determine mechanisms of CCHF transmission.
Cutting-edge immunological tools were developed to understand host responses and support vaccine design. Using Massively Parallel Ribosome Profiling (MPRP), researchers identified hundreds of previously unknown viral open reading frames (ORFs) in CCHFV and other hemorrhagic viruses. These hidden ORFs may play roles in immune evasion and are promising targets for vaccines or antiviral drugs.
Messenger RNA (mRNA) vaccine candidates encoding four CCHFV antigens—nucleocapsid, mutant nucleocapsid, glycoprotein GP38, and deglycosylated GP38—were tested in vivo. All four candidates elicited robust immune.
Single-cell RNA sequencing (scRNA-seq) platforms were refined to profile host immune responses at cellular resolution. These systems support simultaneous analysis of transcriptomes, immune cell repertoires, and antigen specificity. More than 65,000 immune cell transcriptomes have been generated from hemorrhagic virus models, and protocols are being adapted for CCHFV research.
Vector studies progressed through the establishment of tick colonies at partner locations including Amblyomma, Haemaphysalis, and Rhipicephalus tick colonies. Transcriptomic analysis of tick salivary glands is underway to identify molecules that facilitate viral transmission. These efforts lay the groundwork for understanding vector competence and for designing transmission-blocking interventions.
Development of the capability to work with livestock in containment is a primary focus and critical for the National Bio and Agro-Defense Facility (NBAF) standup. BSL3 livestock studies using pathogens of agricultural importance were performed to allow researchers to establish competency at BSL3 before advancing to BSL4. Livestock susceptibility studies progressed through challenge experiments in cattle and swine. Notably, a peer-reviewed study in Nature confirmed that lactating cows exhibit markedly higher susceptibility to HPAI H5N1 infection than calves. This finding underscores the need for facilities that can work with livestock of varying size and ages. In the fall of 2025 ARS scientists will initiate a challenge study with Nairobi sheep disease virus, a tick born BSL3 agent that causes severe disease in livestock. At the BSL4 level an initial infection study in livestock was performed with overseas partners. Samples were collected and will be analyzed in the coming months and will be used to support other collaborative efforts.
Objective 3: Characterize the ecology of CCHFV and enhance surveillance of high-risk regions.
A cornerstone of this project is the investigation of regions known or suspected to harbor CCHFV. Researchers conducted longitudinal field-based sampling in multiple high-risk countries. In East Africa, sampling efforts yielded more than 16,000 ticks collected from livestock, wildlife, and peri-domestic environments. From the first round of sampling in Uganda, 43 of 937 tick samples tested positive for CCHFV, reflecting a 4.6% positivity rate. These samples are currently undergoing virome sequencing, and full-genome assembly is scheduled for completion by December 2025. Additional tick samples (>10,000) remain in cold storage in-country and will be screened in future work.
Microsatellite analysis of Rhipicephalus appendiculatus revealed high gene flow across the Uganda-Kenya cattle corridor, indicating that tick populations are not genetically isolated and are likely moving across borders via livestock trade. Similar analyses for Hyalomma rufipes, a known vector of CCHFV, are underway, with 14 microsatellite markers now validated. These data highlight the role of livestock movement in the cross-border spread of infected ticks and the importance of regional cooperation in vector control.
In the Democratic Republic of Congo (DRC), a nine-province surveillance program collected over 2,000 livestock serum samples. Serological testing in cattle revealed widespread CCHFV IgG positivity, indicating previous exposure. Human sample testing is ongoing. The surveillance network includes commercial farms, village herds, and abattoirs, with paired behavioral and veterinary practice surveys to understand risk factors. A new laboratory was established in Kinshasa with capacity for molecular diagnostics, AI-driven tick identification, and biobanking—strengthening national ownership of surveillance data and enhancing One Health infrastructure.
Additional international surveillance was conducted in Eastern Europe and Türkiye. In Bulgaria and Romania, tick identification was completed, and CCHFV testing is in progress. Serum samples from over 1,600 horses and donkeys were collected across the Balkans, and ticks from migratory birds were screened for CCHFV at a newly established ecological monitoring station outside Istanbul. It is important to note that ticks are only one part of the full life cycle of CCHFV, and the virus may pass through these vectors, to natural and domestic hosts (birds and livestock, respectively), and finally to secondary hosts (humans). In Kosovo, sampling of Hyalomma spp. ticks, agriculture support animals and wildlife has shown high seroprevelance rates in horses and donkeys demonstrating the role multiple species in the maintenance and spread of CCHFV. Ecological models including vector/host dynamics, the environmental conditions under which they occur, to spatiotemporally predict the prevalence of CCHFV under current and future conditions are under development.
In Sierra Leone, researchers completed tick sampling in 37 cattle herds across the country, collecting 5,754 ticks representing multiple genera. Ticks were banked and linked to detailed metadata, including GPS coordinates and animal husbandry practices. In Tanzania, longitudinal studies in 41 herds across four districts were conducted to assess seasonal variation in exposure. Among 823 human participants, 3.4% were seropositive for CCHFV antibodies, with district-level rates ranging from 1.7% to 5.7%. More than 2,800 ticks collected from 380 cattle were pooled and screened by qPCR. Of the 563 pools tested, seven (1.2%) returned suspect-positive results, with Rhipicephalus contributing disproportionately. These finding challenges assumptions that Hyalomma is the sole competent vector and expands the range of tick genera considered relevant for surveillance.
Pilot wildlife surveillance was launched to sample ground-feeding birds, rodents, and lagomorphs in agro-pastoral interfaces. In total, 135 rodents, 140 lagomorphs, and 75 birds were targeted across multiple sites. This work informs future surveillance design in the United States, where similar species may play analogous ecological roles.
Domestically, groundwork was laid to characterize baseline tick biodiversity in U.S. agricultural and peri-urban environments. Infrastructure to support high-throughput data integration was developed to enable real-time modeling of introduction risk and outbreak dynamics. Partnerships were established and local surveillance was initiated to provide a baseline for domestic tick biodiversity and pathogen presence. These data are essential to model potential pathways of introduction, establishment risk, and long-term ecological consequences of CCHFV in U.S. agricultural and peri-urban landscapes.
The combined activities directly support U.S. agriculture by enhancing early warning systems, protecting farm workers, and reducing economic vulnerability. This program is building a framework to prevent, detect, and respond to high-consequence tick-borne viruses before they can threaten American farms and agriculture.
Accomplishments
1. Advanced research tool to support detection of detection of spread and introduction of CCHFV. A suite of molecular and serologic diagnostic tools is being optimized for CCHFV detection. These include LAMP and CRISPR-based assays that are compatible with plasma and serum, peptide-based enzyme-linked immunosorbent assays, and bead-based assay capable of detecting antibodies in livestock, and wildlife populations. Platforms were engineered to function with limited support and will be tested with real-world field specimens. These systems will allow mapping and monitoring of CCHFV and are essential for detecting the virus at ports of entry, abattoirs, and within wildlife-livestock interfaces.
2. New vaccines show promise against high-consequence hemorrhagic fever viruses. Researchers developed and tested four messenger RNA vaccine formulations targeting different proteins of Crimean-Congo Hemorrhagic Fever virus (CCHFV), including the nucleocapsid, mutant nucleocapsid, GP38, and a deglycosylated GP38 variant. All formulations produced robust immune responses in vivo, characterized by both strong T cell and antibody activation. These data provide the foundation for future challenge studies in high-containment settings. This vaccine platform can be rapidly adapted to emerging strains and provides a scalable countermeasure for agricultural and occupational exposure risks.
3. Global investigation identifies new geographic risks and vectors. Researchers conducted large-scale field surveillance in East Africa, Eastern Europe, Central Africa and West Africa, collecting more than 20,000 tick specimens. Analysis is ongoing but positive samples have been identified, including in a tick species not previously recognized as transmitting CCHFV. Using microsatellite population genetics and ecological data, researchers demonstrated that cross-border livestock movement is a major driver of Crimean-Congo Hemorrhagic Fever virus (CCHFV) spread. These results help forecast areas of introduction risk to the United States and support early-warning systems for livestock and wildlife exposure.
4. Development to full life cyle models of Crimean-Congo Hemorrhagic Fever virus (CCHFV). Advanced, full-life cycle models of CCHFV (that include vector, primary/secondary host, and environmental conditions) at ecological hotspots have been developed by ARS researchers (Manhattan, Kansas), and will be trained and tested using the large-scale data collected this year.
5. Field studies define livestock-wildlife-human transmission interfaces. Serologic and vector surveillance was conducted by ARS researchers (Manhattan, Kansas) in livestock and human populations in Tanzania, including ground-feeding birds, rodents, and lagomorphs in agricultural zones adjacent to wildlife parks. Antibodies to Crimean-Congo Hemorrhagic Fever virus (CCHFV) were detected in both cattle and humans, with hot-spot areas showing prevalence rates over five percent. These results provide direct evidence of zoonotic transmission in ecologically vulnerable areas and guide future One Health surveillance programs in the United States and abroad. Similar studies are underway in DRC.
6. Tick colony establishment enables vector competence studies in containment. Colony lines of high-risk tick genera including Amblyomma, Haemaphysalis, and Rhipicephalus were successfully established at partner locations with the help of ARS researchers (Manhattan, Kansas). Other colonies are under development. These colonies provide the critical infrastructure needed to study vector competence, transmission pathways, and virus-host-vector interactions under high biocontainment conditions. This capability is essential for evaluating tick-targeted interventions and mitigation strategies relevant to U.S. agriculture.
7. Hidden viral proteins revealed through advanced ribosome profiling. A novel screening technique—Massively Parallel Ribosome Profiling (MPRP)—has identified over 4,200 previously unrecognized protein-coding regions in 679 human-associated viruses, including high-risk pathogens like Nipah virus. Significantly, some of these hidden proteins are presented by the immune system, offering fresh targets for diagnostics, vaccines, and therapeutics. This matters to USDA because understanding the full set of viral proteins is essential for developing reliable tests and vaccines that detect real infections, not just known protein fragments. Rapidly mapping viral proteins—even in viruses that can’t be grown in labs—helps make sure the U.S. is prepared for emerging and engineered threats, protecting farmers, livestock, and food trade.
8. Real-time data pipelines support global biosurveillance. Researchers in partnership with ARS scientists (Manhattan, Kansas) are developing a digital data pipeline to integrate sequencing, serologic, and ecological metadata to develop data driven models. These platforms enable rapid risk assessment and situational awareness and support the United States Department of Agriculture’s goals of enhancing global preparedness and response. This will allow USDA to prevent, detect and respond to threats to US agriculture and livestock.
9. Deep learning model enhances analysis of liver pathology in hemorrhagic fever models. A convolutional neural network model was trained on digitized liver tissue sections from animals infected with Ebola and Lassa viruses. The model was able to reliably segment hepatic structures such as bile ducts, arteries, and veins with over 99.5 percent specificity. This allows ARS researchers to map pathological changes with greater consistency and less observer bias. The platform will be adapted to Crimean-Congo Hemorrhagic Fever virus models and supports future high-throughput pathology workflows and regulatory decision-making. These models will unlock how the virus produces diseases and facilitate development of countermeasures.
10. Massively parallel ribosome profiling accelerates discovery of viral gene expression. Using Massively Parallel Ribosome Profiling, researchers identified new viral open reading frames across multiple virus families, including Crimean-Congo Hemorrhagic Fever virus. This technique offers high-resolution insights into viral protein production and can reveal novel targets for vaccines or therapeutics. These data provide critical insights into how the virus hijacks host machinery and help prioritize countermeasure development.
11. Mass spectrometry advances tick surveillance. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used to create a library of spectral profiles for medically important tick species in North America by ARS researchers (Manhattan, Kansas). These tools allow for rapid identification of vector species and are being validated for pathogen detection. This approach supports high-throughput tick screening and enhances early detection of vector-borne threats to agriculture.
12. Metagenomics detection of Crimean-Congo Hemorrhagic Fever in waste run off. Minimally invasive, population based investigation of CCHFV was initiated in East and West Africa by ARS researchers (Manhattan, Kansas) using wastewater surveillance and waste from abattoirs. Development of these tools will aid in development of risk based models and increase genomic databases essential to ongoing research efforts focused on understanding transmission, pathogenicity and development of control measures.
13. Technology transfer expands access to tools. Peptide-based diagnostics, data analysis tools, and serologic assays are being shared by ARS researchers (Manhattan, Kansas) with multiple partners to facilitate ongoing research efforts and to facilitate data interpretation across multiple regions. These tools will be critical to early detection of the virus, and allow rapid response to protect US agriculture and livestock.
Review Publications
Hensley, L.E., Bohler, F., Cashman, K., Wilkinson, E., Jonson, J.C., Rosenke, K., Shamblin, J., Honko, A., Shaia, C. 2024. An atlas of gross and histologic lesions and immunohistochemical immunoreactivity during the temporal progression of aerosolized Lassa virus induced hemorrhagic fever in cynomolgus macaques. Frontiers in Cellular and Infection Microbiology. 14. https://doi.org/10.3389/fcimb.2024.1341891.
Malembi, E., Hasivirwe, E., Lusamaki, E., Kalonji, T., Pukuta, E., Hoff, N.A., Bogoch, I., Cevik, M., Gonsalves, G., Hensley, L.E., Low, N., Shaw, S., Schillberg, E., Hunter, M., Lunyanga, L., Linsuke, S., Madinga, J., Kololo, T., Rimoin, A.W., Kindrachuk, J., Mbala-Kingebeni, P., Shongo, R. 2023. Clade I-associated MPXV cases associated with sexual contact in Kwango Province, Democratic Republic of the Congo. Emerging Infectious Diseases. 30(1):172-176. https://doi.org/10.3201/eid3001.231164.
Halwe, N., Cool, K., Breithaupt, A., Schon, J., Trujillo, J., Nooruzzaman, M., Kwon, T., Ahrens, A., Britzke, T., Mcdowell, C.D., Piesche, R., Singh, G., Pinho Dos Reis, V., Kafle, S., Pohlmann, A., Gaudreault, N.N., Corleis, B., Matias-Ferreyra, F.S., Carossino, M., Balasuriya, U., Hensley, L.E., Morozov, I., Covaleda, L.M., Diel, D., Ulrich, L., Hoffmann, D., Beer, M., Richt, J.A. 2024. H5N1 clade 2.3.4.4b dynamics in experimentally infected calves and cows. Nature. https://doi.org/10.1038/s41586-024-08063-y.
Lee, A.W., Liu, K., Lhomme, E., Blie, J., Mccullough, J., Onorato, M.T., Connor, L., Simon, J.K., Dubey, S., Vanrheene, S., Deutsch, J., Owens, A., Morgan, A., Welebob, C., Hyatt, D., Nair, S., Hamze, B., Guindo, O., Sow, S.O., Beavogui, A.H., Leigh, B., Samai, M., Akoo, P., Serry-Bangura, A., Fleck, S., Secka, F., Lowe, B., Watson-Jones, D., Roy, C., Hensley, L.E., Kieh, M., Coller, B.G. 2023. Immunogenicity and vaccine shedding after 1 or 2 doses of rVSV¿G-ZEBOV-GP Ebola Vaccine (ERVEBO®): Results from a Phase 2, randomized, placebo-controlled trial in children and adults. Clinical Infectious Diseases. 78(4):870-879. https://doi.org/10.1093/cid/ciad693.
Normandin, E., Triana, S., Raju, S.S., Lan, T.C., Lagerborg, K., Rudy, M., Adams, G.C., Deruff, K.C., Logue, J., Liu, D., Strebinger, D., Rao, A., Messer, K.S., Sacks, M., Adams, R.D., Janosko, K., Kotliar, D., Shah, R., Crozier, I., Rinn, J.L., Mele, M., Honko, A.N., Zhang, F., Babadi, M., Luban, J., Bennett, R.S., Shalek, A.K., Barkas, N., Lin, A.E., Hensley, L.E., Sabeti, P.C., Siddle, K.J. 2023. Natural history of Ebola virus disease in rhesus monkeys shows viral variant emergence dynamics and tissue-specific host responses. Cell Genomics. 3(12). https://doi.org/10.1016/j.xgen.2023.100440.
Kotliar, D., Raju, S., Tabrizi, S., Odia, I., Goba, A., Momoh, M., Sandi, J., Nari, P., Phelan, E., Tariyal, R., Eromon, P.E., Mehta, S., Robles-Sikisaka, R., Siddle, K.J., Hensley, L.E. et al. 2024. Genome-wide association study identifies human genetic variants associated with fatal outcome from Lassa fever. Nature Microbiology. 9:751-762. https://doi.org/10.1038/s41564-023-01589-3.