Research Project: Rift Valley Fever Pathogenesis and Epidemiology

Location: Foreign Arthropod Borne Animal Disease Research

2025 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 (RVFV) infections, pathogenesis and maintenance in vector and animals hosts continued over the past year. Arthropod-borne viruses must be able to infect and replicate in two disparate hosts (the arthropod vector and the mammalian host) for the natural transmission cycle to occur. While virus infection causes different pathogenicity in mammalian host and insect vectors, little is known about interactions important for these differences. Rho-GTPases are highly conserved proteins found in all eukaryotic organisms and are known to play a crucial role in the replication of several viruses. To examine the role of GTPase in RVFV infection, inhibitors are needed to prevent GTPase activation. A dose response curve was performed for the GTPase inhibitors in several cell lines. This information provided the minimum concentration needed to specifically inhibit the activity of each Rho-GTPase. Last year the activation of Rho GTPases at different times post RVFV infection was determined for both mammalian and insect cells. The information from these two studies will be combined to examine the role of these proteins in RVFV replication and pathogenicity in the disparate hosts. In addition, collaborators in Manitoba, Canada have propagated stocks of RVFV derived from cells of relevant mammalian and arthropod hosts and the purification of mature RVFV particles was optimized. Over the next year, assays will be performed on the purified virus particles to characterize the effects of the mammalian host and insect vector on virus packaging that are important for transmission and pathogenesis of the virus. Together these studies help characterize host, vector and bunyavirus interactions (molecular and cellular) associated with virus infections that are important for virus replication, pathogenesis and transmission and will identify possible targets to interrupt the transmission cycle of RVFV. The central dogma for RVFV transmission has mosquitoes as the primary vector. However, studies have detected RVFV in other arthropods, including biting midges. Thus, an ongoing project is examining the competence of various North American arthropods. For these studies, cells derived from biting midges and ticks were infected with the vaccine strain of RVFV. The permissiveness of the biting midge cell line was examined at two different temperatures representing the temperatures of the insect’s peak activity (27-34°C). Preliminary data demonstrated that temperature may play a role in the permissiveness of midge cells. Conversely, infection of tick cells was examined at a single temperature, with limited success. While arthropods play a critical role in the RVFV transmission cycle, the virus may also be transmitted by direct interaction with infected animals. Current studies are focused on informing the risks associated with non-vector routes of RVFV transmission, such as through consumption or sexual transmission. A few reports suggest that the RVFV may disseminate to the semen of infected mammalian hosts. This raises the question of whether semen is a viable mode of non-vector transmission. To determine the feasibility of RVFV being transmitted through semen, infection of bovine epithelial testis cells and virus stability in bull semen were performed this past year. RVFV replicated in bovine epithelial testis cells over a period of 5 days which suggests a means of maintaining the virus in semen. Progress in assessing the stability of RVFV in semen over the past year included method development for the isolation of viable virus from semen and examining differences in RVFV stability between fresh and frozen semen. Understanding the role of semen in RVFV transmission is important to elucidate the role of this atypical route of the transmission in the maintenance and spread of RVFV. In rural areas where RVFV is endemic, it is common practice for residents to consume raw milk. Field studies in these geographical areas suggest that consumption of unpasteurized milk may contribute to increase RVFV exposure and transmission. Additionally, raw milk is transported to informal markets where residents may purchase milk from other areas where an active outbreak of RVFV may be occurring. Despite the link between milk exposure and RVFV infection, there are still gaps regarding the risk of milk as a non-vector mode of transmission. Researchers from Manhattan, Kansas, in collaboration with researchers from Stanford, California performed a two-center study examining the stability of RVFV in milk. The thermostability of virus in milk from two relevant mammalian hosts (cows and goats) were examined at three different temperatures for 5 days. For both cow and goat’s milk, virus concentration remained stable for up to 96 hours at 4°C and 25°C. At a temperature representing the environmental condition during transport (34°C), infectious virus was no longer detected after 48 hours in both cow’s and goat’s milk. While the virus is more stable in milk at lower temperatures, common pasteurization protocol from endemic areas were effective at killing infectious virus. This data demonstrates that RVFV contaminated milk in the milk supply chain of endemic areas can represent an alternate route of transmission. These studies further demonstrated that viral genomic material was detectable for up to 96 hours in milk, regardless of storage temperature. Thus, milk may act as a non-invasive method for surveillance. As outbreaks of RVFV occur at irregular intervals, it is important to understand how the virus is maintained in nature between epidemics. This was the focus of Objective 2: Identifying factors affecting inter-epidemic cycle and disease emergence caused by Rift Valley fever virus. As part of this objective, epidemiological and ecological factors were examined. The mosquitoes’ internal temperature fluctuates based on its surrounding environmental temperature. The changes in the mosquito’s internal temperature can affect the replication and dissemination of the virus to the salivary glands; thereby altering the ability of the insect to transmit the virus. A collaboration between researchers in Manhattan, Kansas, and Fort Collins, Colorado, examined the effects of temperature on RVFV dissemination in various mosquito species and mosquito determinants associated with any differences. For this study, infected mosquitoes were maintained at one of three target temperatures, representing the lower and upper limits for transmission as determined by modeling from previous published work using global pathogen/mosquito data. Following infection, mosquitoes were dissected at different timepoints post infection to determine the dissemination of RVFV. Following initial analysis, it was determined that the initial virus concentration may influence the dissemination rate. Thus, these studies are being expanded to include a different infectious dose. The need for a deployable tool for rapid diagnostic is important to identify possible infections during the epidemic and inter-epidemic periods. The data obtained from these diagnostic tools will characterize the current RVFV strain and help to determine any shifts that may affect pathogenicity and spread. For this project, a point-of-need molecular assay has been developed and qualified. The qualification of this assay included the sensitivity and specificity to detect RVFV from several matrices representing possible samples collected in the field. These matrices include mosquitoes, serum and milk. In addition, a target approach for obtaining viral sequence using a piece of deployable equipment was also developed using stock and historical samples. A pipeline was created in collaboration with scientists in Manhattan, Kansas, and is currently in transition to the USDA. Over the past year, scientists from Manhattan, Kansas, traveled to endemic areas to evaluate these point-of-need diagnostics and the developed pipeline. As part of this travel, the scientist from Manhattan, Kansas, trained Kenyan collaborators on the equipment and tested field samples. Preliminary data from these studies demonstrated, for the first time, RVFV infection of a threatened wildlife species in Kenya. In addition to the efficacy of these point-of-need diagnostics, this data will provide information regarding the role of a new wildlife species in the maintenance and transmission of RVFV. These deployable diagnostics allow for rapid detection needed to inform the response to the outbreak. In addition, the data obtained from these assays will provide additional information to improve transmission models and risk assessments. Lastly, ARS scientists from Manhattan, Kansas, organized RVFV gap analysis workshop comprising of 40 scientists from 20 different countries. The participants had RVFV expertise in the following categories: virology, diagnostics, vaccines, epidemiology and vector control. This workshop produced productive discussion and identification of several knowledge gaps that can be used to guide future projects that will benefit the stakeholders and protect the United States agriculture.

Accomplishments
1. Udderly stable: Rift Valley fever virus stability in raw cow and goat milk. Rift Valley fever virus (RVFV) is a zoonotic disease that causes devastating epidemics that affect humans and livestock (e.g. cattle, sheep and goats) with tremendous economic impact during an outbreak. Multiple investigations have implicated milk as a possible mode of RVFV transmission. In endemic areas, raw milk is transported to informal markets where residents may purchase milk from areas of an active RVFV outbreak. This study demonstrated that the virus is stable in both cow and goat milk for over 24 hours at a representative temperature during transport. However, standard pasteurization techniques from endemic countries prove efficacious for inactivating the virus. This data demonstrates that RVFV contaminated milk in the milk supply chain of endemic areas can represent an alternate route of transmission. These studies further demonstrated that genetic material of the virus was detectable for up to 96 hours in milk, regardless of storage temperature. Thus, milk may act as a non-invasive method for surveillance. Surveillance is key for the early detection of RVFV, which can limit disease progression, reducing the impact to both human and agricultural health.

Review Publications
Azerigyik, F.A., Cagle, S., Wilson, W.C., Mitzel, D.N., Kading, R.C. 2025. The temperature-associated effects of Rift Valley fever virus infections in mosquitoes and climate-driven epidemics: A review. Viruses. 17(2). Article 217. https://doi.org/10.3390/v17020217.
Landers, R.S., Hodgson, P., Puckette, M.C., Chaki, S.P., Wilson, W.C., Higgs, S., Zuelke, K.A. 2025. Retrospective case study of the impacts of multiple One Health oriented biocontainment research facilities during the SARS-CoV-2 pandemic. One Health. 20. Article 101061. https://doi.org/10.1016/j.onehlt.2025.101061.
Dawes, B.E., De La Mota-Peynad, A.M., Rezende, I.M., Buyukcangaz, E.K., Harvey, A.M., Gerkin, K.N., Winter, C., Bayrau, B., Mitzel, D.N., Waggoner, J.J., Pinsky, B.A., Wilson, W.C., Labeaud, A.D. 2025. Rift Valley fever virus remains infectious in milk stored in a wide range of temperatures. Journal of Infectious Diseases. Article jiaf060. https://doi.org/10.1093/infdis/jiaf060.