Research Project: Viral Ecology of Henipaviruses in Endemic Settings and Intervention Strategies to Prevent their Spread to Domestic Animals

Location: Zoonotic and Emerging Disease Research

2025 Annual Report

Objectives
Objective 1. Characterize the ecology of henipahviruses with a focus on the One-Health concept. - Conduct the molecular characterization of new and emerging henipahviruses, including phylogenetics, and network analysis. - Examine known or emerging henipahviruses that may have an impact on animal agriculture, including their potential impact on public health. Objective 2. Elucidate the host-pathogen interactions of henipahviruses infections. - Investigate virus-specific factors and viral molecular markers associated with infectivity, pathogenicity and transmissibility of henipahviruses in susceptible animal species including virus tissue tropism and replication. - Investigate host-specific factors associated with the infectivity, pathogenicity and transmissibility in different animal species. - Characterize the innate and adaptive immune response to henipahviruses infections in animal models that are either susceptible, tolerant, or resistant to infection. Objective 3. Develop surveillance strategies and early warning systems for henipahviruses. - Improve surveillance strategies to detect henipaviruses in high-risk countries. - Establish a formal laboratory network for henipavirus surveillance that includes standardized specimen collection, laboratory testing scheme, quality control, specimen referral and accreditation.

Approach
Henipahviruses are members of the family Paramyxoviridae, order Mononegavirales. The name henipavirus was recommended for the genus of both Hendra virus and Nipah virus. Henipaviruses have a large host range, unlike other members of the Paramyxoviridae, which generally have a very narrow host range. The cell attachment protein, unlike many other members for the paramyxovirus subfamily, does not have haemagglutinating activity and as a consequence does not bind sialic acid on the surface of cells. The natural reservoir of the henipaviruses are fruit bats mainly from the genus Pteropus (flying foxes). Nucleic acid and antibody signatures of exposure to Nipah virus or Nipah-like viruses has been documented in a diversity of bat species across the globe. The threat for a natural introduction of henipaviruses in the United States is low, but there is significant concern that henipaviruses could be used for nefarious purposes to harm agriculture and people. Both Hendra virus and Nipah virus are on the HHS and USDA list of overlap Select Agents and Toxins. Henipaviruses are listed as APHIS Tier 3 high-consequence foreign animal diseases and pests. Henipaviruses are promiscuous in their ability to cause severe morbidity in several animal species, including people, and human infections result in a very high mortality rate. The mortality rate associated with Nipah virus infections in pigs has been reported to be approximately 2.5% in adult pigs – high morbidity, but low mortality. Mortality rates in humans however are significantly higher and range from 40% (Malaysia) to 75% (up to 100%) in Bangladesh. The animal reservoir includes several species of bats, and henipaviruses may thus be readily available in these wildlife reservoirs.

Progress Report
Henipaviruses, are high-consequence zoonotic pathogens with significant implications for public health, livestock production, and national security. This year’s work represents significant strides across the three objectives of the project—ecological surveillance, host-pathogen interaction, and diagnostic preparedness—and positions USDA to protect food and farming systems through science. Objective 1: Characterize the ecology of henipaviruses with a focus on the One-Health concept. Field studies in Puerto Rico and Indonesia are clarifying critical ecological and behavioral determinants of virus maintenance, transmission, and spillover risk. In Puerto Rico, researchers conducted over 100 environmental air sampling events and sampled more than 2,500 bats across multiple seasons. These efforts uncovered genomic material from four novel paramyxoviruses and validated air-based viral surveillance as a scalable, non-invasive approach that can be used across environments—from bat caves to animal barns. In Indonesia, ARS scientists participated in a longitudinal market survey across 29 wildlife markets. Researchers documented the persistent sale of live bats for medicinal use, often slaughtered on-site. This work is revealing regional, demographic, and social correlates with higher bat sales. Markets were found to have peak bat volumes on weekends, indicating social behavior patterns that influence spillover risk. These data will be incorporated into USDA and interagency risk models to inform import policies, surveillance targeting, and trade negotiations. In Bangladesh, ARS-supported fieldwork targeted viral reservoirs in peri-urban and agricultural settings. Over 1,300 samples were collected from 444 animals across six host types, including bats, shrews, and rodents. Sample types included oral swabs, urine, serum, and rectal swabs. This diverse sample matrix enables viral genome discovery and serological screening for exposure to Nipah virus. Bioinformatic pipelines are processing the data for detection of known and novel pathogens. This effort supports USDA’s risk mapping, global threat detection, and preparedness frameworks. In parallel, novel trapping tools were deployed to capture larger mammals, pending final permitting. These studies will expand USDA's capacity to model intermediate host ecology and transmission dynamics. Researchers are also coordinating with local diagnostic laboratories to validate molecular and serological tools in-country, enhancing regional diagnostic capacity and USDA's international response infrastructure. To support broader biosecurity objectives, ARS researchers contributed to the surveillance of novel henipa-like viruses and mpox virus lineages across West and Central Africa. In Nigeria, ARS scientists supported efforts to apply agnostic sequencing to wildlife and livestock, identifying three novel henipa-like viruses from fruit bats and rodents. These discoveries are under genomic and serological follow-up. These findings directly inform USDA’s models for zoonotic emergence in agroecological interfaces. ARS scientists participated in research efforts to address Disease X concerns in the Democratic Republic of Congo (DRC) and Nigeria. No new henipaviruses were identified but evidence of other pathogens of interest to USDA were identified and highlighted the need to enhance surveillance for agriculture and cross over pathogens. These actions validated our capacity to mobilize U.S. agricultural science in response to unpredictable threats and provided invaluable training opportunities for partner countries, helping to create a global buffer that slows pathogen emergence before U.S. exposure. Collectively, this research strengthens USDA’s One Health posture. Our approach integrates ecological surveillance, host-pathogen biology, diagnostic innovation, and global partnerships to proactively defend U.S. agriculture and livestock from high-consequence viral threats. Recently, there has been increasing concern about these and other diseases entering the United States either accidently or purposefully via legal and illegal wildlife (bushmeat) trade to the United States either directly from countries of origin or indirectly from Europe. This work ensures every measure has been taken to understand how these pathogens are able to move and spread through the environment, to inform prevention and mitigation preparedness. As the risk of emerging zoonoses accelerates with urbanization, and global trade, USDA’s role in early detection and response becomes increasingly vital to protect U.S. agriculture and trade. Objective 2: Elucidate the host-pathogen interactions of henipaviruses infections. Development of the capability to work with livestock in containment is a primary focus and critical to the standup of USDA’s Biosafety level four (BSL4) facility. Livestock studies were initiated in BSL3 with pathogens of agriculture importance to meet this need. Establishing competency at lower BSL is essential to worker safety and biosecurity. Studies of high path avian influenza (HPAI) in cattle confirmed that lactating cows exhibit markedly higher susceptibility to HPAI H5N1 infection than calves. This finding underscores the importance of physiological state in disease dynamics. Additional work at BSL-3 with swine evaluated infection dynamics of MPOX and avian influenza. Under other collaborative efforts Marburg infection of swine was initiated. This pilot study laid the foundation for eventual work with livestock of varying size and age to accurately characterize the disease and risk in agriculture species to BSL4 agents. To investigate the viral and host-specific factors that influence henipavirus infectivity, pathogenicity, and transmission, including tissue tropism and immune responses across animal species with varying susceptibility ARS scientists have partnered with other active BSL4 labs to work with available models. As part of these efforts a severe disease model was used to perform a sequential sampling to evaluate viral dissemination, immune evasion, and organ pathology. In a follow up experiment alternate routes of challenge were used to determine if the route of exposure would impact disease severity. Tissues from these models are undergoing a detailed examination including single-cell RNA sequencing to define host responses at a cellular level, identifying critical pathways involved in severe disease. In parallel, the team is applying massively parallel ribosome profiling (MPRP) to uncover hidden viral proteins expressed during infection. This approach allows for unbiased discovery of non-canonical viral proteins and host-virus interactions, offering novel targets for diagnostics and therapeutics. In addition to work in animal models a clustered regularly interspaced short palindromic repeats(CRISPR) based library of cells with individual genes knocked out is also being utilized to identify host factors critical for pathogenesis and viral replication. The MPRP and CRISPR based libraries will be utilized to characterize novel henipaviruses. Objective 3: Develop surveillance strategies and early warning systems for henipaviruses. To advance diagnostic capability and pathogen characterization, ARS researchers are developing multiple field-deployable molecular diagnostics including loop-mediated isothermal amplification (LAMP) and CRISPR-based assays, CRISPR- and LAMP-based molecular assays tailored for field deployment. These assays were optimized for animal biospecimens (e.g., plasma, capillary blood), and successfully lyophilized into stable, transportable reaction beads for use with the low-complexity microfluidic diagnostic (LCMD) platform. This innovation reduces user steps, eliminates cold chain dependence, and allows for deployment in austere environments. A biochemical filtration method was integrated into the sample prep workflow to enhance matrix tolerance. Parallel efforts in serology involved the development of short-peptide libraries (pan-Henipa, pan-Nairo) and phage display and species agnostic bead based serologic assays. These tools were validated using sera from experimentally infected animals, convalescent human patients, and wildlife species. The resulting epitope data identified high-confidence diagnostic targets for differential serodiagnosis of Nipah virus and related pathogens, now being incorporated into multiplex non-invasive population-based detection is being piloted through genomic surveillance of wastewater and abattoir waste. These efforts, if successful, will be evaluated for utility of other sources of waste run-off including gutters from wild-life markets. The inclusion of AI tools further expands analytic power. The deep learning system used to annotate liver damage across hemorrhagic fever viruses will be adapted to henipavirus animal models. These tools accelerate insights into pathogenesis and vaccine efficacy, shortening the time from field detection to policy decision. Ultimately, this program not only builds scientific capability but also protects rural livelihoods and national food systems. By equipping USDA with validated diagnostics, research assays, serologic tools, host-pathogen data, and pre-approved partnerships, this work ensures that the U.S. is not only reactive but strategically positioned to prevent agricultural crises before they emerge. These efforts strengthen farmer resilience, enable informed trade negotiations, and enhance national biosecurity policy frameworks. This program continues to demonstrate that investment in foundational science with global scope pays dividends for U.S. preparedness, market stability, and agricultural leadership. Continued support will ensure USDA remains at the forefront of zoonotic threat mitigation in an increasingly interconnected world.

Accomplishments
1. Nipah virus livestock model developed for high-risk scenario testing. A severe disease model was developed to study Nipah virus infection. The model was not in livestock but enables viral pathogenesis across different infection routes and development of assays and practices for use at the National Bio and Agro-Defense Facility (NBAF). Tissues from these studies are being analyzed with single-cell RNA sequencing to define cellular responses and massively parallel ribosome profiling to discover hidden viral proteins. These data support vaccine and diagnostic development. Farmers and livestock producers benefit from understanding how the virus spreads and causes disease in animals.

2. Rapid diagnostics enable field detection of emerging threats. ARS researchers in Manhattan, Kansas with partners are developing molecular assays for henipavirus detection that can be deployed in the field without cold chain requirements. Using CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and LAMP (Loop-mediated Isothermal Amplification), they created freeze-dried tests usable on portable diagnostic platforms. These tools are designed to detect infection in animals quickly and cheaply, supporting the capacity to contain outbreaks before they spread.

3. Risk of bat -borne viruses discovered in U.S. Territories. ARS researchers in Manhattan, Kansas, with partners collected over 2,500 bats in Puerto Rico Genomic analysis revealed multiple novel paramyxoviruses and other previously unknown viruses. These findings help assess which viruses are evolving in regions with agricultural trade links to the U.S. Early detection of novel viruses abroad protects farmers at home.

4. Risk of bat -borne viruses discovered in West and Central Africa. In Nigeria and the Democratic Republic of the Congo, ARS researchers in Manhattan, Kansas, with partners are exploring unknown morbidity and mortality in livestock. Historical samples are being evaluated for previous exposure to henipaviruses. These findings help assess which viruses are evolving in regions with agricultural trade links to the U.S. Early detection of novel viruses abroad protects farmers at home.

5. Long-term bat trade monitoring identifies persistent spillover risk. A wildlife market study in Indonesia performed by ARS researchers in Manhattan, Kansas, with partners identified seasonal and demographic patterns tied to bat volume and market activity. The findings inform trade policy and disease risk models, strengthening border biosecurity and protecting U.S. livestock producers.

6. Henipavirus antibodies detected using peptide-based serologic tests. Short peptide libraries representing key viral proteins were developed by ARS researchers in Manhattan, Kansas, with partners, enabling antibody detection from exposed animals and humans. These tools work across species and reduce false positives by avoiding whole-virus cross-reactivity. This innovation helps determine which regions or species have been exposed to high-consequence pathogens.

7. Air-based biosurveillance validated as scalable non-invasive approach. A pilot study study performed by ARS researchers in Manhattan, Kansas, with partners in Puerto Rico showed that air sampling could detect paramyxoviruses in bat caves and agricultural areas. Four new viruses were detected, and environmental variables like humidity and sampling height were assessed. Air surveillance may enable early warning systems for emerging diseases in U.S. livestock regions.

8. Investigation of potential spillovers in emerging zoonotic hotspots. Teams including ARS researchers from Manhattan, Kansas, investigated unknown illness clusters in the DRC. Samples were collected from using a One Health investigation approach. No new novel pathogens were discovered but other agriculture threats were discovered. These efforts build a global early warning network that benefits U.S. agriculture by identifying threats before they reach American farms.

9. Surveillance extended to small mammals and ticks at livestock markets. Over 300 animals and 300 ticks were sampled by ARS researchers in Manhattan, Kansas, with partners in Nigerian livestock markets in 2025. Samples are being sequenced to identify known and emerging zoonotic viruses. Findings inform understanding of pathogen transmission in high-risk trade hubs. Livestock producers and trade stakeholders care about these findings due to their impact on disease spread and market access.

10. 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 by a team including ARS researchers in Manhattan, Kansas. 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.

11. Novel host-pathogen tools improve livestock preparedness. Artificial intelligence (AI) tools were developed by ARS researchers in Manhattan, Kansas, with partners to analyze liver damage and inflammation in animal models. These tools help quantify disease severity and evaluate treatments in a standardized way. As tissues for henipaviruses are available, the tools will be utilized to investigate disease pathogenesis. Farmers benefit from understanding which interventions reduce mortality and productivity losses.

12. High-risk animal populations identified through market surveillance. ARS researchers in Manhattan, Kansas, with partners conducted systematic surveillance of livestock markets in high-risk regions. Sampling of live animals revealed a subset of species and market conditions that increase spillover risk of zoonotic pathogens. These findings help guide future USDA surveillance activities, ensuring resources are focused where risks are highest.

13. Portable serology platforms deployed in-country. Field-validated, species-agnostic serologic assays were deployed by ARS researchers in Manhattan, Kansas, with partners to local diagnostic labs in Africa and Asia. These tests allowed rapid detection of prior exposure to henipaviruses and other high-consequence viruses in animals and humans. This supports USDA goals of early detection and global situational awareness.

14. Veterinary biosafety training delivered to frontline personnel. In partnership with ARS researchers in Manhattan, Kansas, training was provided to local veterinarians, public health workers, and wildlife officers. These sessions built capacity for early outbreak recognition and safe specimen handling. U.S. agricultural interests benefit from a trained global workforce that can identify threats before they cross borders.

15. Multiplex viral genome detection through targeted enrichment. ARS researchers in Manhattan, Kansas, with partners applied VirCapSeq, a targeted viral enrichment platform, to diverse animal and environmental samples. This approach improved the sensitivity of viral detection in pooled and low-concentration samples. It supports comprehensive pathogen discovery without requiring large sequencing budgets.

16. Regional partnerships expanded for zoonotic disease modeling. Collaborative work in Asia, Africa, and U.S. territories expanded the network of regional institutions contributing to zoonotic disease modeling. These relationships allow USDA to access near real-time ecological data to inform outbreak prediction and agricultural risk management.

Review Publications
Vakaniaki, E.H., Merritt, S., Linsuke, S., Malembi, E., Muyembe, F., Lunyanga, L., Mayuma, A., Kwete, P., Kalonji, T., Madinga, J., Lebreton, M., Nakoune, E., Kalthan, E., Shang, S., Nwobegahay, J., Ehiakhamen, O., Dibongue, E., Kakou, J., Erima, B., Byarugaba, D., Kinzie, P., Mebwa, F., Baelongandi, F., Kayolo, A., Nabugobe, P., Mwambia, D., Malekani, J., Nguete, B., Kabamba, J., Kaba, D., Hensley, L.E., Kindrachuk, J., Liesenborghs, L., Shongo, R.L., Muyembe-Tamfum, J., Hoff, N.A., Rimoin, A.W., Mbala-Kingebeni, P. 2025. Establishment of a regional mpox surveillance network in Central Africa: Shared experiences in an endemic region. Acarology International Congress Proceedings. https://doi.org/10.1186/s41256-025-00408-y.
Halbrook, M., Makangara-Cigolo, J.C., Merritt, S., Hoff, N.A., Lisenborghs, L., Hensley, L.E., Vercauteren, K., Mbala-Kingebeni, P., Rimoin, A.W., Kindrachuk, J. 2024. Mpox in Central Africa: Complex epidemiology requires a constellation approach. npj Viruses. https://doi.org/10.1038/s44298-024-00081-x.
Lo, M.K., Jain, S., Davis, K.A., Sorvillo, T.E., Welch, S.R., Coleman-Mccray, J.D., Chatterjee, P., Hotard, A.L., O'Neal, T., Flint, M., Ai, H., Albarino, C.G., Spengler, J.R., Montgomery, J.M., Spiropoulou, C.F. 2024. Optimization of Bangladesh and Malaysian genotype recombinant reporter nipah viruses for in vitro antiviral screening and in vivo disease modeling. Antiviral Research. 231. https://doi.org/10.1016/j.antiviral.2024.106013.
Hensley, L.E., Kinganda-Lusamaki, E., Amuri-Aziza, A., Fernandez-Nunez, N., Makangara-Cigolo, J., Pratt, C., Hasivirwe Vakaniaki, E., Hoff, N.A., Lukanda-Ndelemo, G., Akil-Bandali, P., Sabiti Nundu, S., Mulopo-Mukanya, N., Ngimba, M., Modadra-Madakpa, B., Diavita, R., Paku-Tshambu, P., Pukuta-Simbu, E., Merritt, S., O'Toole, A., Low, N., Nkuba-Ndaye, A., Kavunga-Membo, H., Shongo Lushima, R., Liesenborghs, L., Wawina-Bokalanga, T., Vercauteren, K., Mukadi-Bamuleka, D., Subissi, L., Muyembe-Tamfum, J., Kindrachuk, J., Ayouba, A., Rambaut, A., Delaporte, E., Tessema, S., D'Ortenzio, E., Rimoin, A.W., Mbala-Kingebeni, P., Peeters, M., Ahuka-Mundeke, S. 2025. Clade I mpox virus genomic diversity in the Democratic Republic of the Congo, 2018-2024: Predominance of zoonotic transmission. Cell. 188(1). Article 4-14.e6. https://doi.org/10.1016/j.cell.2024.10.017.
Merritt, S., Halbrook, M., Kompany, J., Chandrasekaran, P., Smith, O.A., Hoff, N.A., Tambu, M., Martin, S.A., Wong, T., Jarra, A., Barrall, A.L., Musene, K., Beya, M., Orr, R., Myers, T., Macgill, T., Hensley, L.E., Muyembe-Tamfum, J., Kaba, D., Berry, I.M., Mbala-Kingbeni, P., Lehrer, A.T., Rimoin, A.W. 2025. Comparison of EBOV GP IgG antibody reactivity: Results from two immunoassays in the Democratic Republic of the Congo. Journal of Virological Methods. 336. https://doi.org/10.1016/j.jviromet.2025.115154.
Hensley, L.E., Klena, J.D., Deboer, J.T., Montgomery, J.M., Mbala, P., Moses, M.E., Knapek, K.J., Olinger, G.G. 2024. Laboratory needs for research response. In: Sorenson, R.A., editor. Principles and Practice of Emergency Research Response. Springer. https://doi.org/10.1007/978-3-031-48408-7_11.
Hensley, L.E., Hasivirwe Vakaniaki, E., Kacita, C., Kinganda-Lusamaki, E., O'Toole, A., Wawina-Bokalanga, T., Mukadi-Bamuleka, D., Amuri-Aziza, A., Malyamungu-Bubala, N., Mweshi-Kumbana, F., Mutimbwa-Mambo, L., Belesi-Siangoli, F., Mujula, Y., Parker, E., Muswamba-Kayembe, P., Nundu, S.S., Lushima, R.S., Makangara-Cigolo, J., Mulopo-Mukanya, N., Pukuta-Simbu, E., Akil-Bandali, P., Kavunga, H., Abdramane, O., Brosius, I., Bangwen, E., Vercauteren, K., Sam-Agudu, N.A., Mills, E.J., Tshiani-Mbaya, O., Hoff, N.A., Rimoin, A.W., Kindrachuk, J., Baxter, C., De Oliveira, T., Ayouba, A., Peeters, M., Delaporte, E., Shuka-Mundeke, S., Mohr, E.L., Sullivan, N.J., Muyembe-Tamfum, J., Nachega, J.B., Rambaut, A., Liesenborghs, L., Mbala-Kingebeni, P. 2024. Sustained human outbreak of a new MPXV clade I lineage in eastern Democratic Republic of the Congo. Nature Medicine. 30:2791-2795. https://doi.org/10.1038/s41591-024-03130-3.
Mantlo, E., Trujillo, J.D., Gaudreault, N.N., Morozov, I., Lewis, C.E., Matias-Ferreyra, F., Mcdowell, C., Bold, D., Kwon, T., Cool, K., Balaraman, V., Madden, D., Artiaga, B., Souza-Neto, J., Doty, J.B., Carossino, M., Balasuriya, U., Wilson, W.C., Osterrieder, N., Hensley, L.E., Richt, J.A. 2024. Experimental inoculation of pigs with monkeypox virus results in productive infection and transmission to sentinels. Emerging Microbes & Infections. 13. Article 2352434. https://doi.org/10.1080/22221751.2024.2352434.
Vakaniaki, E.H., Kinganda-Lusamaki, E., Merritt, S., Kasongo, F., Malembi, E., Lunyanga, L., Linsuke, S., Halbrook, M., Kalthan, E., Pukuta, E., Aziza, A.A., Cigolo, J.M., Lumembe, R., Kabamba, G., Anta, Y., Bolunza, P., Kanda, I., Ngazobo, R., Kalonji, T., Nsio, J., Matoka, P., Mwamba, D., Ngandu, C., Shaw, S.Y., Shongo, R., Madinga, J., Boum, Y., Liesenborghs, L., Delaporte, E., Ayouba, A., Low, N., Mundeke, S.A., Hensley, L.E., Tamfum, J.M., Nakoune, E., Peeters, M., Hoff, N.A., Kindrachuk, J., Rimoin, A.W., Mbala-Kingebeni, P. 2024. Presumed transmission of 2 distinct monkeypox virus variants from Central African Republic to Democratic Republic of the Congo. Emerging Infectious Diseases. 30(10):2128-2134. https://doi.org/10.3201/eid3010.241118.