Research Project: Rift Valley Fever Pathogenesis, Epidemiology, and Control Measures

Location: Foreign Arthropod Borne Animal Disease Research

2021 Annual Report

Objectives
1. Identify factors associated with Bunyaviridae (Rift Valley Fever virus) infections, pathogenesis, and maintenance in vector and animal hosts. Subobjective 1A: Identify viral molecular determinants of virulence and mechanisms of viral pathogenesis in relevant animal hosts associated with arthropod-transmitted virus. Subobjective 1B: Characterize host, vector and bunyavirus interactions (molecular and cellular) associated with virus infection. 2. Identify epidemiological and ecological factors affecting the inter-epidemic cycle and disease emergence caused Bunyaviridae (Rift Valley Fever virus). Subobjective 2A: Develop means to detect and characterize emergent arboviral diseases and use these data to generate models that predict future outbreaks. Subobjective 2B: Identify the biotic and abiotic factors that favor establishment of emerging arboviruses and use these data to generate models that predict future outbreaks.

Approach
The potential introduction of Rift Valley fever (RVF) virus (RVFV) is the most significant arthropod-borne animal disease threat to U.S. livestock according to the USDA-APHIS National Veterinary Stockpile (NVS) Steering Committee. A number of challenges exist for the control and prevention of RVF in the areas of disease surveillance, diagnostics, vaccines and vector control. RVFV is the third biological threat agent on the NVS Steering Committee’s priority list for generation and stockpiling of countermeasures for diagnosis, vaccination, and insect control. Understanding the epidemiological factors affecting disease outbreak and the interepizootic maintenance of RVFV is necessary for the development of appropriate countermeasures strategies. This includes the ability to detect and characterize emergent viruses since RVFV is an RNA virus and could evolve to adapt to a new environment. Also, the proposed research will identify determinants of RVFV infection, pathogenesis and maintenance in mammalian and insect vector hosts. Information derived from these studies will also provide a better vaccine evaluation challenge model. Vaccine formulations will be developed to improve immunogenicity, onset of immunity and stability to provide better response to outbreaks and prevent RVFV epizootics. The overall goals of this project are to utilize the unit’s unique multidisciplinary expertise to fill knowledge gaps about the interepidemic cycle of RVFV and provide the tools necessary for detecting, controlling and eradicating RVFV should it be introduced into the U.S.

Progress Report
This is the final report for project 3020-32000-014-00D entitled “Rift Valley Fever Pathogenesis, Epidemiology, and Control Measures” which ends on 11/15/2021. Since the start of this project, progress was made on all subobjectives contained within the projects two main objectives. This section describes the advancements made during the full-life of this project. Rift Valley fever (RVF) virus (RVFV) is an exotic, arthropod-borne, zoonotic pathogen that poses a significant threat to U.S. livestock if introduced. Subobjective 1A is directed towards identifying viral determinants of virulence and pathogenesis. RVFV genome consists of three segments that can be exchanged between different genetic types and potentially closely related viruses, thereby producing new viruses. This has a significant impact on virus evolution, virulence and ability to spread. The rate at which this exchange of segments, called reassortment, can occur is important to know since the attenuated live vaccine is the only conditionally approved vaccine in the United States. Different molecular tools, such as, reverse-transcriptase polymerase chain reaction (RT-PCR), plaque purification and sequencing were developed to evaluate reassortment rate. Virus reassortment rate was investigated in cell-culture and in sheep, a target hosts species. In addition, a reverse genetic system was established in the laboratory. Synthetic viruses representing two wildtype RVFV strains were rescued using the reverse genetics system and the pathogenicity of the synthetic virus was compared to wildtype virus in sheep. This system helps to elucidates the roles of different viral proteins. Understanding the roles of these proteins is critical in predicting the impact of newly emerged genetic strains of RVFV. Arthropod-borne viruses are maintained in nature through complex interactions between the virus, the invertebrate vector, the vertebrate host and the environment. Understanding these interactions can reveal novel strategies for developing countermeasures against virus transmission and pathogenesis (Sub-objective 1B). Previous studies examining RVFV infection in livestock animals demonstrated strain differences in the clinical pathology. Sequence analysis indicated that genetic variation among the virus population within the inoculum contributed to the different pathological outcome. Further analysis demonstrated that the virus population was dependent on the host origin: mosquito vs vertebrate host. In addition, differences in the glycosylation pattern of RVFV virions coming from insect cells were identified when compared to mammalian cells. These differences could have physiological implications which effect the virus ability to infect and replicate in the different host. Current studies are examining the effects of protein host origin on RVFV replication. In addition, during feeding, the mosquito not only injects the pathogen but also its saliva. The vector’s saliva has been shown to play a role in the transmission of several pathogens. Saliva enhancement of RVFV infection was examined by utilizing saliva from Culex tarsalis, a competent mosquito species, and primary bovine macrophage cells. Immunological markers effected by the presence of virus with and without mosquito saliva was identified. In addition to identifying factors associated with virus infections, pathogenesis and maintenance, other studies related to this project examined and developed possible medical countermeasures. These studies included development of an efficacious subunit RVFV vaccine which was patented and licensed to a commercial company for development (Animal and Plant Health Inspection Service license pending). Currently, no antiviral treatment exists for RFVF. Therefore, studies were performed to screen a panel of potential drugs. This screening revealed two possible candidates for antiviral treatment. Studies are ongoing to understand the mechanism and to identify other potential candidates. This project also developed and identified tools for virus detection. This included improvements made to tools for distinguishing between infected and vaccinated animals. In addition, a multiplex fluorescence microsphere immunoassay (FMIA) was developed and evaluated the detection of different antibodies against multiple RVFV proteins. This assay was evaluated from samples collected in Kenya, demonstrating its usefulness as a diagnostic and surveillance tool in an endemic country. A RVF competitive enzyme linked immunosorbent assay was also developed and packaged commercially. These assays allow for safe generation of serological assays for antibodies against RVFV and could provide early warning to the introduction of the virus into non-endemic areas such as the United States. These assays could also help identify animals infected during the inter-epidemic cycle. The information collected from these assays can help model epidemiological and ecological factors important for virus maintenance between epidemics as well as disease emergence. To further develop epidemiology models, an invasive mosquito project, a citizen science-based crowd sourced method for mosquito collection was established with community members and local experts, as part of Sub-objective 2A. New insect traps were developed and distributed to zoos to help collect mosquitoes for testing. Since the start of the project, the partnership has expanded into California and increased the number of long-term collection locations beyond the citizen scientists. Mosquitoes from this effort were shipped from around the country. As part of Sub-objective 2B, biotic and abiotic factors that favor establishment of arboviruses were identified and these data generated models that predict future outbreaks. One ecological factor important for modelling is environmental temperature. Environmental temperature is a key factor in virus replication and maintenance during the inter-epidemic cycle. Studies utilizing two mosquito species, demonstrated the temperature effects on the ability of RVFV to infect, disseminate and be transmitted was species dependent. As part of this Sub-objective, methods to differentiate true signal from noise in the data were used to make predictive models. These models used the abundance and distribution of the Asian tiger mosquito, a common disease vector, based on environmental factors (temperature and precipitation). The location of the mosquito collection and an arthropod-borne virus (Dengue) was mapped, and the entomological risk estimated per month globally. This established a predictive model that can be utilized for multiple pathogens, such as RVFV.

Accomplishments
1. Host origin plays a role in virus replication. Arthropod-borne viruses, such as Rift Valley fever virus (RVFV), must replicate within two distinct hosts (the arthropod and mammalian host). Each host places distinct pressures on the virus which results in different patterns of mutations and post translational modifications such as the addition of molecules to virus proteins. These differences could have physiological implications which effect the virus ability to infect and replicate in the different host. ARS scientists at Manhattan, Kansas in collaboration with Kansas State University, demonstrated that the addition of sugars (glycosylation) to RVFV proteins on the surface of the virus particle are different among the virus particles coming from mammalian and those coming from insect cells. Furthermore, replication kinetic studies in a single cell line demonstrate that the origin of the virus played a role in the replication efficiency of RVFV. These results could be due to the different glycosylation patterns association with the disparate hosts. Understanding the role of these host origin modifications could lead to the identification of a virus determinants important for virus replication and pathogenesis.

2. Efficacy of a Rift Valley fever virus subunit vaccine in cattle. Rift Valley fever is a disease that causes significant morbidity and mortality in livestock, especially sheep and cattle. The causative agent, Rift Valley fever virus (RVFV), is an arthropod-born virus. Due to the presence of the many competent vectors of the virus in non-endemic area, the virus has enormous potential for continuing its transboundary spread. However, there is no fully licensed vaccine suitable for use in non-endemic areas. Previously, ARS scientists at Manhattan, Kansas, in collaboration with Kansas State University demonstrated the efficacy of a patented recombinant subunit vaccine in sheep. This group of scientists recently expanded on this research to examine the efficacy of the subunit vaccine formulations in cattle. These studies demonstrated that the subunit vaccine protected cattle from Rift Valley fever. This study supports the notion, that the subunit vaccine platform can prevent and control RVFV infections in target animals, making it a promising and safe strategy for control and prevention of an outbreak in non-endemic areas.

3. Transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by blood-feeding insects. At the beginning of the coronavirus disease 2019 (COVID-19) pandemic, there was no direct evidence of virus transmission by blood-feeding insects. Several ARS scientists at Manhattan, Kansas, in collaboration with Kansas State University have contributed to the understanding of SARS-CoV-2 transmission by blood-feeding insects. Scientist fed two North American species of mosquitoes and one midge species on SARS-CoV-2 spiked blood. It was determined that these insects did not support virus replication and therefore do not pose a risk for transmission to animals or humans. This was the first study to examine the role of midges in the transmission of the virus and helps to answer the questions regarding the role of insects in the transmission of the newly emergent disease.

4. Entomological risk prediction platform. Researchers in Manhattan, Kansas, in collaboration with Kansas State University, developed risk prediction models that can estimate virus transmission based on weather and climate increasing insect vector abundance before the onset of disease. Public health officials can use the models to forecast cases (2-3 days in the future based on daily case reports) and reposition resources if cases are increasing or decreasing. The model is robust enough to use with any case data and not just vector-borne disease. Furthermore, if insufficient case data is present in an area due to gaps in surveillance or reporting, the model can use disease cases in similar surrounding geographic areas to fill in the missing data temporally or spatially. Lastly, the model can estimate disease risk anywhere in the world based on the historic reports of cases, vector insect presence, climate, and weather. The model provides a high, medium, or low risk output to help public health and military planners get the proper equipment to travelers on overseas deployments.

Review Publications
Ragan, I., Bradshaw, K., Upreti, D., Odendaal, L., Richt, J., Trujillo, J.D., Wilson, W.C., Davis, A.S. 2019. Rift Valley fever viral RNA detection by in situ hybridization in formalin-fixed, paraffin-embedded tissues. Vector-Borne and Zoonotic Diseases. 19(7):553-556. https://doi.org/10.1089/vbz.2018.2383.
Kroeker, A.L., Babiuk, S., Pickering, B.S., Richt, J.A., Wilson, W.C. 2020. Livestock challenge models of Rift Valley fever for agricultural vaccine testing. Frontiers in Veterinary Science. 7:239. https://doi.org/10.3389/fvets.2020.00238.
Gaudreault, N.N., Madden, D., Wilson, W.C., Trujillo, J.D., Richt, J.A. 2020. African swine fever virus: An emerging DNA arbovirus. Frontiers in Veterinary Science. 7:215. https://doi.org/10.3389/fvets.2020.00215.
Oliveira, A., Cohnstaedt, L.W., Cernicchiaro, N. 2021. Unbiased approaches for reviewing entomology literature: A systematized review. Annals of the Entomological Society of America. 114(2):229-246. https://doi.org/10.1093/aesa/saaa058.
Smith, M., Schirtzinger, E.E., Wilson, W.C., Davis, A. 2019. Rift Valley fever virus: Propagation, quantification, and storage. Current Protocols in Microbiology. 55(1):e92. https://doi.org/10.1002/cpmc.92.
Gaudreault, N., Trujillo, J., Carossino, M., Meekins, D., Madden, D., Indran, S., Morozov, I., Bold, D., Balaraman, V., Kwong, T., Roman-Sosa, G., Artiaga, B., Cool, K., Garcia-Sastre, A., Ma, W., Wilson, W.C. 2020. SARS-CoV-2 infection, disease and transmission in domestic cats. Emerging Infectious Diseases. 9(1):2322-2332. https://doi.org/10.1080/22221751.2020.1833687.
Gaudreault, N.N., Morozov, I., Trujillo, J.D., Meekins, D.A., Carossino, M., Madden, D.W., Cool, K., Libanori-Artiaga, B., McDowell, C., Bold, D., Ma, W., Henningson, J., Balasuriya, U.B., Wilson, W.C., Garcia-Sastre, A., Richt, J.A. 2021. Experimental re-infected cats do not transmit SARS-CoV-2. Emerging Microbes & Infections. 10(1):638-650. https://doi.org/10.1080/22221751.2021.1902753.
Endalew, A., Faburay, B., Trujillo, J.D., Gaudreault, N.N., Davis, A., Shivanna, V., Sunwoo, S., Ma, W., Drolet, B.S., McVey, D.S., Morozov, I., Wilson, W.C., Richt, J. 2019. Immunogenicity and efficacy of Schmallenberg virus envelope glycoprotein subunit vaccines. Journal of Veterinary Science. 20(6):e58. https://doi.org/10.4142/jvs.2019.20.e58.
Wilson, W.C., Mitzel, D.N., Savini, G., Zientara, S., Richt, J.A. 2020. Editorial: Emerging arboviruses. Frontiers in Veterinary Science. 7:593872. https://doi.org/10.3389/fvets.2020.593872.