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- Cohnstaedt, Lee
- Wilson, William
- Mitzel, Dana
- Miesner, Tracy
- Mire, Chad
- 2023
- 2022
- Publications
- Animal Health (103)

Research Project: Japanese Encephalitis Virus Prevention and Mitigation Strategies **Location:
                            Foreign Arthropod Borne Animal Disease Research** 2023 Annual Report **Objectives**
                                

OBJECTIVE 1: Identify factors associated with Flavivirus infections, pathogenesis, and maintenance in vectors and animal hosts to inform prevention and mitigation strategies.
• Identify factors associated with JEV maintenance in relevant insect vectors.
• Characterize susceptibility, pathogenesis, and clinical disease of JEV in swine.
• Characterize vector-virus-host interactions associated with JEV transmission.
Sub-objective 1.A. Evaluate the ability of an emerging JEV genotype to infect and replicate in North American domestic swine and mosquito vectors.
Sub-objective 1.B. Investigate potential roles of North American feral swine and biting midges in JEV transmission. **Approach**
                                

Japanese encephalitis virus (JEV) is a zoonotic arthropod-borne pathogen native to Asia and the Pacific Rim, where it is a significant cause of reproductive and neonatal loss in swine and severe encephalitis and death in humans. JEV is transmitted to vertebrate hosts by infected mosquito vectors and has demonstrated an ability to emerge in new geographic regions that contain competent vectors and susceptible hosts. JEV does not currently circulate in the United States (U.S.); however, the risk of its introduction has been assessed as high (Oliveira et al. 2020). Significant research gaps exist regarding U.S. vulnerability following an introduction of JEV, including the range of native vectors and hosts capable of sustaining transmission and whether U.S. mosquitoes and livestock are vulnerable to emerging genotypes of JEV. This project will address these gaps by evaluating the ability of an emerging JEV genotype to infect and replicate in domestic swine and mosquitoes and by investigating the potential roles of previously uncharacterized wildlife hosts and insect vectors in JEV transmission. These studies will use in vitro and in vivo infection models to investigate the effects of wild-type JE viruses on insect vectors and mammalian hosts (Objective 1). Additionally, next generation sequencing and genomic analyses will be used to study vector-virus-host interactions to determine effects that hosts and vectors have on virus populations. The knowledge gained will be used to inform risk assessments and predictive models and help identify target points to guide diagnostic development, surveillance programs, and control strategies. Together, these measures will help strengthen the U.S. disease prevention and response framework for rapidly stopping foreign animal disease incursions to protect the health and profitability of U.S. livestock. **Progress Report**
                                

Objective 1. Significant progress was made on both sub-objectives. In support of Sub-objective 1A, seven strains of Japanese encephalitis virus (JEV), representing four of the five known genotypes have been acquired for study. Replication kinetics studies for wildtype JEV genotypes I-III (GI-III) were initiated in cell lines from four different mosquito species. Two experimental replicates have been completed for each genotype in the four cell lines. Data analysis to compare replication of the viruses in the different mosquito cell lines and between the different genotypes is ongoing. These studies will provide data regarding the potential for the different genotypes to replicate in various North American mosquitoes; thereby providing input to strengthen risk assessments.  
In studies focused on animal hosts, North American domestic swine were experimentally challenged with a JEV GII virus for the first time. Samples were collected to determine virus replication, seroconversion, and transcriptomics. Analysis is ongoing to measure the amount and kinetics of virus distribution and shedding, virus pathogenesis, and development of successful immune responses. This information will help scientists assess whether GII viruses may be a threat to U.S. swine in the event of a JEV incursion. In a separate project, swine were challenged with an attenuated vaccine strain of JEV to generate reference sera for immune studies and diagnostic development. The reference sera were determined to have high antibody titers as measured by plaque reduction neutralization tests. These samples will serve as a useful resource for validating protein expression and other projects moving forward. In a third project, ARS scientists demonstrated experimental feeding of two species of Culex mosquitoes on pigs for the first time. Mosquitoes of both species fed to repletion with probing sites evident on both shaved and haired skin of nursery piglets. These results have implications for mosquito-borne infectious diseases, as well as for pest control efforts by producers. Samples were also collected to investigate whether immune and inflammatory responses were induced by the mosquito feedings.  
In support of Sub-objective 1B, collaborators at Texas Tech University collected samples from feral swine which will be tested for flaviviruses to establish a baseline for surveillance and to pre-screen the samples for planned collaborative research projects. JEV can infect a wide range of animals, and the full host range is not known. West Nile virus, a closely related flavivirus, has been known to infect North American white-tailed deer (WTD). Additionally, WTD have been known to act as reservoirs for other zoonotic viruses, notably including SARS-CoV-2. ARS scientists performed two independent experiments to examine the in vitro susceptibility of two types of primary WTD cells to an attenuated strain of JEV. Virus was observed to replicate in both cell types, in some cases to high titers, indicating that additional studies of the susceptibility of WTD to JEV may be warranted to determine whether they could have a role in the maintenance of JEV in nature. Additional investigations into the host range for JEV in North American wildlife were performed by collaborators at Colorado State University. Pythons and leopard frogs were challenged with the four genotypes of JEV, and blood was collected to detect viremia at regular intervals post-inoculation. Viremia was detected at low levels for multiple animals, but the magnitude did not appear compatible with reservoir or amplifying host function. Clinical disease was not observed in any snake, however apparent neurologic disease was observed in two frogs and is being followed up with additional testing. Samples have also been collected for viral genome analysis.
In collaboration with researchers at The Pennsylvania State University, chimeric viruses were created using mosquito-borne and tick-borne flaviviruses to identify viral determinants important for flavivirus infection and replication within different vector/host species. Initial experiments demonstrated that exchanging the structural proteins did not prevent entry, suggesting that non-structural proteins may play a more critical role of flavivirus infection in mammalian hosts. In addition, the data from these studies will identify possible vaccine candidates for emerging flaviviruses. As part of separate programs with two commercial partners, major JEV proteins have been expressed for use towards the development of new vaccines and diagnostic tests. A third commercial partner has developed new vaccine constructs which have undergone initial in vivo testing.    
In collaboration with researchers from Kansas State University, a workflow has been developed to characterize virus population genetics of JEV. Flaviviruses exist as heterogenous populations composed of groups or individual viruses with distinct genetics. Historically, these populations were represented by a single consensus sequence. Consensus sequences do not accurately describe the population as they are created by determining the most frequent nucleotide at each site. The importance of evaluating the individual components of viral populations is demonstrated when a minority virus quickly adapts to a selective pressure or environmental change allowing that virus to replicate more efficiently and enhancing the spread of the virus. A member of the viral population capable of evading the selective pressure is difficult to determine from a consensus sequence. **Accomplishments**
                                

1. Realtime vector-borne disease and mosquito vector forecasting. Predicting Insect Contact and Transmission Using historical Entomological and Environmental data (PICTUREE) is an outbreak forecasting tool developed by Kansas State University engineers and ARS researchers in Manhattan, Kansas. The tool was evaluated to forecast case data during dengue outbreaks in Nepal and Bangladesh and mosquito abundance and distributions in Southern California. The case forecasting tool takes human case data and uses various algorithms (ensemble Kalman filters, particle filters, and deep neural networks) to calculate the number of cases one, two, and three weeks into the future. This allows health care and emergency response workers to preposition materials and allocate equipment to areas. It also informs policy makers if the outbreak is continuing to grow or if it is declining. Ultimately it may be used to determine the cost effectiveness of management methods based on actual cases versus predicted cases. Similarly, the algorithms are being used to predict mosquito abundance in California for three mosquito control districts in the absence of viruses. This confirms managements actions and improves their mosquito control efficiency. 2. Construction of Japanese encephalitis virus (JEV) peptide library. Japanese encephalitis is a significant cause of reproductive loss and neonatal death in swine in Asia and the Pacific Rim. Cytotoxic T-lymphocytes (CTL) are cells that are important for cell-mediated immune responses to viral infections. Little is known about the role that CTL play during JEV infections of swine. To find regions of JEV proteins that can induce CTL in swine, Kansas State University scientists used the vaccine strain of JEV to construct and validate expression constructs for the 10 JEV genes that comprise the virus proteome as part of a collaborative project with ARS researchers in Manhattan, Kansas. They screened the expressed proteins to identify peptide motifs that bind strongly to alleles of the Swine Leukocyte Antigen [SLA]-I complex region of the pig genome and identified multiple JEV proteins that contain putative CTL epitopes. They used the information to generate a library of 120 synthetic peptides which will be used to evaluate JEV antigen-specific T-cell responses. This resource will enable them to better understand swine immune responses and will inform the design of vaccines that can stimulate host CTL responses to prevent and mitigate disease. **Review Publications**
                            *Yi, C., Vajdi, A., Ferdousi, T., Cohnstaedt, L.W., Scoglio, C. 2023. PICTUREE—Aedes: A web application for dengue data visualization and case prediction. Pathogens. 12(6):771. https://doi.org/10.3390/pathogens12060771.*
                                *Holcomb, K.M., Mathis, S., Staples, J.E., Fischer, M., Barker, C.M., Beard, C.B., Nett, R.J., Keyel, A.C., Marcantonio, M., Childs, M.L., Gorris, M.E., Rochlin, I., Hamins-Puertolas, M., Ray, E.L., Uelmen, J.A., Defelice, N., Freedman, A.S., Hollingsworth, B.D., Das, P., Osthus, D., Humphreys Jr, J.M., Nova, N., Mordecai, E.A., Cohnstaedt, L.W., Kirk, D., Kramer, L., Harris, M.J., Kain, M.P., Reed, E.M., Johansson, M.A. 2023. Evaluation of an open forecasting challenge to assess skill of West Nile virus neuroinvasive disease prediction. Parasites & Vectors. 16(1):11. https://doi.org/10.1186/s13071-022-05630-y.*
                                *Rochlin, I., White, G., Reissen, N., Swanson, D.A., Cohnstaedt, L.W., Chura, M., Healy, K., Faraji, A. 2022. Laboratory evaluation of sugar alcohols for control of mosquitoes and other medically important flies. Scientific Reports. 12(1). Article 13763. https://doi.org/10.1038/s41598-022-15825-z.*
                                *Ewing, R.D., Brokesh, B., Shults, P.T., Cohnstaedt, L.W. 2023. Are you still using 6-volt batteries for your insect traps? American Mosquito Control Association. 39(1):61-64. https://doi.org/10.2987/22-7061.*
                                *Cernicchiaro, N., Oliveira, A., Cohnstaedt, L.W. 2022. Epidemiology of infectious diseases. In: McVey, S., Kennedy, M., Chengappa, M.M., Wilkes, R., editors. Veterinary Microbiology. 4th edition. Hoboken, NJ: John Wiley and Sons. p. 818-828. https://doi.org/10.1002/9781119650836.ch72.*


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