Research Project: Rift Valley Fever Pathogenesis and Epidemiology

Location: Foreign Arthropod Borne Animal Disease Research

2024 Annual Report

Objectives
Objective 1: Identify factors associated with Bunyaviridae (Rift Valley Fever virus) infections, pathogenesis and maintenance in vector and animal hosts. • Characterize host, vector and bunyavirus interactions (molecular and cellular) associated with virus infection. • Characterize the host immune response in susceptible animal hosts and determine influences that determine clinical outcomes during primary RVFV infections. • Further identify competent mosquito species in the United States that can amplify and spread RVFV, and also investigate the capacity for U.S. culicoides and ticks to become competent vectors. • Identify mosquito species that transmit RVFV transovarially. • Determine the risk of transmitting RVFV through unpasteurized milk consumption. • Determine the risk of transmitting RVFV through semen. • Determine the infectious period of RVF virus in cattle and sheep (the period of viremia sufficiently high to result in infection of mosquitoes) and use these data to improve epidemiological models. Sub-objective 1.A: Identify cellular factors important for replication and pathogenesis in the vector and the mammalian host. Sub-objective 1.B: Characterize host immune response and determine influences that effect clinical outcomes during primary RVFV infections in susceptible hosts. Sub-objective 1.C: Competence of North American arthropods to RVFV. Sub-objective 1.D: Identify the ability of mosquitoes to vertically transmit RVFV to the next generation. Sub-objective 1.E: Assessment of risks associate with non-vector routes of RVFV transmission. Objective 2: Identify factors affecting the inter-epidemic cycle and disease emergence caused by Rift Valley Fever virus. • Identify epidemiological and ecological factors affecting the inter-epidemic cycle. • Develop means to detect and characterize emergent arboviral diseases and use these data to generate models that predict future outbreaks. • Determine the infectious period of RVF virus in cattle and sheep (the period of viremia sufficiently high to result in infection of mosquitoes) and use these data to improve epidemiological models. Sub-objective 2.A: Examine the effects of temperature on the extrinsic incubation period of RVFV in various mosquito species. Sub-objective 2.B: Develop deployable tools for rapid diagnostics of RFVF. Sub-objective 2.C: Determination of viremic titers needed for transmission of RVFV to mosquitoes. Objective 3: Develop intervention strategies to effectively control Rift Valley Fever virus and contain disease outbreaks. • Evaluate and improve existing and new diagnostic tests and testing strategies for RVF virus surveillance, detection, and recovery from disease outbreaks. • Evaluate existing vaccine platforms and develop strategies designed to rapidly control and prevent RVF virus outbreaks. Sub-objective 3.A: Evaluation and development of immune-based diagnostic tests for RVFV. Sub-objective 3.B: Develop and evaluate a vaccine platform to control and prevent RVF virus outbreaks.

Approach
Rift Valley fever (RVF) is a long-recognized disease of domestic livestock in Africa caused by the arthropod-borne virus, Rift Valley Fever virus (RVFV). RVFV is a zoonotic pathogen present on the World Organisation for Animal Health (WOAH) list of notifiable animal diseases of concern and the World Health Organization priority disease list. RVFV is a significant threat to U.S. livestock due to the presence of naïve host populations and competent vectors. RVFV transmission is dependent on complex interactions involving virus, arthropod vector, mammalian host, and environment. Despite the importance of this disease, many elements for RVFV replication, pathogenesis, transmission, and disease epidemiology are poorly understood. To address these gaps, this study has three objectives: 1). Identify factors associated with Rift Valley Fever virus infections, pathogenesis and maintenance in vector and animal hosts, 2) Identify factors affecting the inter-epidemic cycle and disease emergence caused by Rift Valley Fever Virus, and 3) Develop intervention strategies to effectively control Rift Valley Fever virus and contain disease outbreaks. Objectives 1 and 2 will be addressed by examining different aspects of the maintenance and transmission of RVFV. These include examining the roles of North American arthropods as primary and auxiliary vectors in virus transmission, assessing the ability of vertical transmission by mosquitoes, and the stability of RVFV in milk and semen as a non-vector route of transmission. In addition, the effects of temperature on RVFV extrinsic incubation period will be examined. The host response will also be examined as part of objective 1. The importance of GTPases in the regulation of virus replication in the disparate hosts will be examined in a set of studies. An additional study will examine the influence of RVFV Gn and Gc antigens on anti-RVFV neutralizing antibodies in animals with different clinical outcomes. Objectives 2 and 3 will be addressed by examining surveillance and control strategies. For control strategies, existing vaccine platforms will be examined through designing and characterizing a RVFV vaccine vector based on the recombinant Vesicular Stomatitis Virus (rVSV) vaccine platform expressing the Gn and Gc RVFV antigens. As part of the diagnostics and surveillance, both molecular and immunological assays will be developed and tested. Some of the assays will be developed as deployable diagnostic tools. These tools will provide data on virus emergence and will assist in surveillance, development of risk assessments, and predictive modeling.

Progress Report
Rift Valley fever virus (RVFV) is a mosquito-borne virus that is transmitted to livestock through the bite of an infected mosquito. Little is known about the virus-vector-host interactions important for the transmission and survival of the virus in nature. To gain insight into these interactions, several projects supporting Objective 1: Identify factors associated with Bunyaviridae (Rift Valley fever virus) infections, pathogenesis, and maintenance in vector and animal hosts continued over the past year. As an arthropod-borne pathogen, RVFV must be able to replicate and transmit between two very different and distinct systems: the arthropod vector and the mammalian host. Rho-GTPases are molecules that play an important role in cell migration and remodeling of the cell structure and thus play an important role in virus replication and are common within several disparate systems. To elucidate how this common pathway effects the replication and pathogenesis of RVFV in the disparate hosts, the activation of Rho GTPases at different times post Rift Valley fever virus infection were performed for both mammalian and insect cells. Rho-Activated GTPases were examined by capturing effector protein that binds specifically to activated GTPases. This type of assay allows for the detection of the activated molecules in infected and uninfected cells. These studies will elucidate how a common pathway effects the replication and pathogenesis in two different hosts. Collaborators in Manitoba, Canada, have optimized virus amplification in various mosquito cell lines and have now been able to obtain large batches of viruses generated from multiple representative insect and mammalian cells. These samples will be used over the next year to characterize the effects of the mammalian and insect host on virus protein packaging. Lastly, studies were initiated that will examine the role of mosquito salivary proteins in the transmission of arboviruses and how environmental changes and infections will affects the protein expression of mosquito salvia. Together these studies help to characterize host, vector and bunyavirus interactions (molecular and cellular) associated with virus infections that are important for virus replication, pathogenesis, and transmission. Thereby, identifying possible targets to interrupt the replication and transmission cycle of the RVFV. Historical serum samples have been identified that can be used in future studies to examine the host immune response in susceptible animal hosts. These studies will determine if specific immune response influence clinical outcome during primary RVFV infection. In rural areas where Rift Valley fever is endemic, it is common for the residents to drink unpasteurized milk. Data analysis from field studies in endemic areas suggest that consumption of raw milk may contribute to increased RVFV exposure and transmission of the virus. To examine the possibility of milk consumption being an alternative mode of transmission, collaborators in Stanford, California, performed a comprehensive literature study. For this study, data on the demographics and milk exposure factors associated with RVFV exposure, basic mechanistic models of transmission, age, location, raw milk consumption, and ruminant ownership were collected. As part of this study, other viruses were identified that exhibit comparable transmission and may serve as potential models. Researchers from Stanford, California, in collaboration with researchers from Manhattan, Kansas performed a two-center study examining the stability of RVFV in milk. The thermostability of milk from two relevant mammalian hosts were examined at various temperatures for 5 days. In addition, the efficacy of common pasteurization protocol from endemic areas was examined. These data will demonstrate how long infectious virus may remain in raw milk as well as determine the possibility of using this milk as an alternative matrix for surveillance. The maintenance of virus during the long period between epidemics, known as the inter-epidemic period, is not well understood. During this time, infection has been detected from mosquitoes, animals and humans which suggest that RVFV is maintained by low-level transmission. Three main studies are ongoing to accomplish Objective 2: Identify factors affecting the inter-epidemic cycle and disease emergence caused by Rift Valley fever virus. Transmission of arthropod-borne viruses, such as RVFV, are greatly influenced by temperature. The mosquito’s internal temperature varies as a consequence of the environmental temperature. Thus, the first study examines the effect of temperature on vector competence. Researchers in Fort Collins, Colorado, completed the examination of the infection and dissemination of RVFV in Culex tarsalis at two temperatures and have initiated studies on a third temperature. As part of this study, the researchers developed a computer program to initiate ways of measuring innate immune gene expression during RVFV in Culex tarsalis mosquitoes. RNA from collected sample is being stored for further transcriptomics studies. Surveillance and identification of RVFV from different mammalian hosts and vectors is important in understanding the key factors in the low-level transmission of RVFV during the inter-epidemic cycle. Thus, a field deployable assay for RVFV is being verified with positive controls and historical samples. In addition, a bioinformatic pipeline for the sequence assembly of RVFV is being developed. The bioinformatic pipeline is being further tested with lab samples. Little is known regarding the infectious period of the mammalian host and the concentration of virus in the mammalian blood needed for virus transmission to the mosquito vector during a blood-meal. A transmission study utilizing mosquito-sheep-mosquito transmission model was completed. This study examines the different stages of a complete transmission cycle since it utilizes infected mosquitoes to inoculate the sheep. At different timepoints, serum was collected from the sheep to examine the concentration of virus in the blood and uninfected mosquitoes were fed on sheep to determine when transmission may occur. This study will provide information on the infectious period and titer needed to infect mosquitoes. In addition, this study also provides information regarding 1) RVFV pathogenesis in sheep when inoculated via a needle versus inoculation by an RVFV infected mosquito (natural infection) 2) identify differences in virus population genetics at each stage of the transmission cycle between sheep and mosquito 3) assess reassortment efficiency in sheep after co-infecting with two RVFV strains. Over the past year, scientists from Manhattan, Kansas, have traveled to endemic areas to evaluate and improve existing and new diagnostic tests. Samples were collected that allowed for testing of lateral flow assay against animal serum. These results provided feedback to the collaborator on appropriate modifications needed to improve the assay. Similar tests have been performed for RVFV enzyme linked immunosorbent assays. These studies help to support Objective 3: Develop intervention strategies to effectively control Rift Valley fever virus and contain disease outbreaks. In addition, as part of Objective 3, several foreign agreements were established to collect various samples including mosquitoes and mammalian serum. Over the past year over 800 serum samples were collected from relevant animals’ species throughout Kenya. These samples will not only be utilized to test new technology and diagnostics but will also be utilized in surveillance. The areas for collection where specifically chosen to inform predictive models regarding climate change and how encroachment of livestock into wild areas effect arbovirus emergence.

Accomplishments
1. Rapid point of care diagnostic for Rift Valley fever virus. The mosquito-borne Rift Valley fever virus (RVFV) causes major agricultural and public health problems in Africa and is considered a potential agro-bioterrorism agent with limited countermeasures. A rapid, and sensitive molecular method for point of care (POC) or point of need (PON) that can be deployed to field applications was established by ARS researchers in Manhattan, KS, in collaboration with Kansas State University scientists. The strategy involves detection of the RVFV genome on a portable, touch screen instrument. The system sensitivity and specificity were tested in the laboratory then evaluated in the two endemic African countries using ruminant sera. The system provides a reliable, sensitive, and specific point of need tool for the detection of genomic RVFV. This new tool can be used in diagnostic, surveillance and epidemiological applications.

2. Rift Valley fever virus surveillance at Kenyan abattoir. ARS researchers surveyed two urban abattoirs and nearby mosquitoes during predicted ideal conditions for a disease outbreak. Although no mosquitoes tested positive for Rift Valley fever virus (RVFV) using a lateral flow assay, 8.5% of the 923 animals tested were positive for antibodies to RVFV. The research demonstrates the potential for using slaughterhouse samples for Rift Valley fever surveillance.

3. Rift Valley fever virus reassortment in sheep. A mosquito transmitted viral disease in ruminant livestock and humans called Rift Valley fever (RVF) is caused by a virus of the same name. The virus is composed of three genome segments which can be exchanged with different strains of the virus in a process called reassortment. Researchers in Manhattan, Kansas, examined the efficiency of this process in sheep co-infected with different strains of the RVF virus. The results indicated that this process is inefficient in sheep. The knowledge learned from these studies on reassortment is important for understanding the dynamics of RVFV evolution.

Review Publications
Balaraman, V., Indran, S.V., Meekins, D., Jakkula, L.U., Liu, H., Hays, M.P., Li, Y., Souza-Neto, J.A., Gaudreault, N.N., Hardwidge, P.R., Wilson, W.C., Weber, F., Richt, J.A. 2023. Identification of host factors for Rift Valley fever phlebovirus. Viruses. 15(11). Article 2251. https://doi.org/10.3390/v15112251.
Balaraman, V., Indran, S.V., Kim, I., Trujillo, J.D., Meekins, D.A., Shivanna, V., Zajac, M.D., Urbaniak, K., Morozov, I., Sunwoo, S., Faburay, B., Osterrieder, K., Gaudreault, N., Wilson, W.C., Richt, J.A. 2024. Rift Valley fever phlebovirus reassortment study in sheep. Viruses. 16(6). Article 880. https://doi.org/10.3390/v16060880.
Gerken, K.N., Owuor, K.O., Ndenga, B.N., Wambua, S., Winter, C., Chemutai, S., Omukuti, R., Arabu, D., Miring’U, I., Wilson, W.C., Mutuku, F., Waggoner, J.J., Pinksy, B., Bosire, C., Labeaud, A.D. 2024. Expanding understanding of urban Rift Valley fever risk and associated vector ecology at slaughterhouses in Kisumu, Kenya. Pathogens. 13(6). Article 488. https://doi.org/10.3390/pathogens13060488.
Cool, K., Gaudreault, N.N., Trujillo, J.D., Morozoz, I., McDowell, C., Bold, D., Souza-Neto, J., Matias-Ferrerya, F., Retallick, J., Singh, G., Schotsaert, M., Carossino, M., Balasuriya, U.B., Wilson, W.C. 2023. Experimental co-infection of calves with SARS-CoV-2 Delta and Omicron variants of concern. Emerging Microbes & Infections. 13(1). Article 2281356. https://doi.org/10.1080/22221751.2023.2281356.
Olajiga, O.M., Jameson, S.B., Carter, B.H., Wesson, D.M., Mitzel, D.N., Londono-Renteria, B. 2024. Artificial feeding system for vector-borne disease studies. Biology. 13(3). Article 188. https://doi.org/10.3390/biology13030188.
Cooper, A.M., Jameson, S.B., Pickens, V., Osborne, C., Backus, E.A., Silver, K., Mitzel, D.N. 2023. An electropenetrography waveform library for the probing and ingestion behavior of Culex tarsalis on human hands. Insect Science. 31(4):1165-1186. https://doi.org/10.1111/1744-7917.13292.
Gibson, S., Noronha, L., Tubbs, H., Cohnstaedt, L., Wilson, W., Mire, C., Mitzel, D., Anyamba, A., Rostal, M., Linthicum, K. 2023. The increasing threat of Rift Valley fever virus globalization: strategic guidance for protection and preparation. Journal of Medical Entomology. 60(6):1197-1213. https://doi.org/10.1093/jme/tjad113.
Norton, A.E., Ewing, R.D., Tilley, M., Whitworth, J., Cohnstaedt, L.W. 2023. Fatal food: Silver-coated grain particles display larvicidal activity in Culex quinquefasciatus. ACS Agricultural Science and Technology. 8(37):33437-33443. https://doi.org/10.1021/acsomega.3c03210.
Kimble, J.B., Noronha, L.E., Trujillo, J., Mitzel, D.N., Richt, J., Wilson, W.C. 2024. In: Hayes, T., Gopal, V., editors. Veterinary Clinics of North America. Philadelphia, PA: Elsevier. p. 293-304. https://doi.org/10.1016/j.cvfa.2024.01.004.
Trujillo, J.D., Wilson, W.C., Craig, A., Van Den Bergh, C., Wang, T., Thompson, P.N., Swanepoel, R., Morozoz, I., Richt, J. 2024. Rift Valley fever virus M and L genome segment detection: A comparison of field deployable reverse transcription insulated isothermal PCR (RT-iiPCR) and laboratory based multiplex reverse transcriptase real-time PCR. Journal of Clinical Microbiology. 63(3). https://doi.org/10.1128/jcm.00430-23.