Identify factors associated with Flavivirus infections, pathogenesis, and maintenance in vectors and animal hosts to inform prevention and mitigation strategies including identifying factors associated with JEV maintenance in relevant mosquito vectors; characterizing susceptibility, pathogenesis, and clinical disease of JEV in domestic pigs; and characterizing vector-host interactions with JEV transmission. Subobjective 1A: Identify factors associated with JEV maintenance in relevant mosquito vectors. Subobjective 1B: Characterize susceptibility, pathogenesis, and clinical disease of JEV in domestic pigs. Subobjective 1C: Characterize vector-host interactions associated with JEV transmission.
Japanese encephalitis virus (JEV) is an arthropod-borne virus (arbovirus) endemic to Asia, where it is the most important cause of viral encephalitis in humans and a significant cause of reproductive and neonatal loss in swine. JEV transmission does not currently occur in the U.S.; however, North American mosquitoes have recently been demonstrated to be competent virus vectors. JEV is closely related to the West Nile Virus, a formerly foreign arbovirus that was introduced into the U.S. in 1999 and quickly became established across most of the country. Pigs are considered to be primary amplifying hosts for JEV; infected pigs are capable of replicating the virus to high titers and are thought to fuel outbreaks by serving as sources of virus for mosquitoes. Although JEV has been studied for 7 decades, considerable knowledge gaps exist regarding its transmission, particularly in the pig-mosquito segment of its cycle. The goal of this project is to better understand mammalian host (pig) and vector (mosquito) factors that permit transmission of JEV in order to identify actions to break the transmission cycle and develop new detection and prevention tools. These gains will support not only livestock health and food security, but also the protection of human health.
This is the final report for the expiring project 3022-32000-023-000D, which was active from June 2018 to May 2022 (previously as 3020-32000-014-000D). This project has been superseded by project 3022-32000-025-000D, which is also entitled “Japanese Encephalitis Virus Prevention and Mitigation Strategies.” Please see that report for additional information about recent progress and accomplishments. Progress was made on the project’s single objective, including understanding Japanese encephalitis virus (JEV) maintenance in relevant mosquito vectors. Work with wildtype JEV is strictly controlled and limited to biosafety level-3 (BSL-3) containment labs. An attenuated strain of JEV was used to develop a biosafety level-2 (BSL-2) surrogate system for in vitro studies of JEV in mosquito cells, which has enabled the investigation of virus replication in lower biocontainment settings than wildtype JEV. JEV is known to grow in some mosquito cells; however, the cell line typically used is not representative of the predominate vectors for the virus, Culex spp. mosquitoes. To determine the ability of JEV to replicate in a more relevant cell line, the attenuated strain of JEV was used to infect two Culex cell lines derived from mosquito species found in the U.S. In one of the cell lines, which was recently developed by ARS scientists in Manhattan, Kansas, replication studies demonstrated that the attenuated JEV strain grew to high titers without causing any noticeable detrimental effects to the cells. These data were the first to show that Culex cell lines are susceptible to infection with the attenuated strain of JEV and also demonstrated that the new Culex cell is line relevant for production of virus stocks and for use in investigating the virus-vector interaction important for replication and maintenance in mosquito vectors, thus laying a foundation for future studies of pathogenesis and maintenance in North American mosquitoes. A new wildtype JEV strain was also acquired and used to infect two species of North American Culex mosquitoes in a BSL-3 level arthropod containment laboratory (ACL-3) for the first time. The results showed that the virus was capable of disseminating to the salivary glands of the mosquitoes, suggesting that these mosquito species can support the replication and transmission of this JEV strain to mammalian hosts/reservoirs if introduced into the U.S. Because the transmission cycle of JEV requires the virus to replicate in disparate hosts such as mammals and insect vectors, studies were also performed to examine the role of the origin of the stock virus (mammalian or mosquito) on virus infection, replication and dissemination in mosquitoes. Replication in the different hosts (insect vs. vertebrate) places different evolutionary pressures on the virus. How these pressures effect the fitness for infection in mosquitoes is unknown. The samples generated from the infected mosquitoes and mammalian host cells were also collected for genetic analysis. Together, these studies will help identify viral determinates important in the replication and transmission of JEV. Progress was made towards understanding JEV maintenance in porcine hosts. New assay systems were developed to measure the impact of viral infections on porcine innate immune cells using a macrophage cell line originally developed by a collaborating ARS scientist in Clay Center, Nebraska. Assays were adapted and applied to develop a framework to study the cellular mechanisms by which arthropod-borne viral infections kill porcine innate immune cells. This was combined with measurements of immune system gene expression to better characterize virus impacts and mechanisms in the immune systems of infected pigs. A BSL-2 surrogate system using attenuated JEV was also established for porcine cells. Dose-response and replication kinetic studies were performed in the macrophage cell line as well as cell lines that originated from other pig tissues that are known, or hypothesized, to be susceptible to JEV infection. Several of the cell lines produced virus at high levels, but experienced variable amounts of cell death, and in some cases none. These results help inform our understanding of pathogenesis in the porcine host by indicating which organs and cells may be more susceptible to infection. This work also established an in vitro infection to facilitate molecular and cellular studies of JEV infection. Additionally, these findings helped identify cell types to take forward into future studies and to use for the growth of virus stocks for other infection studies. This cell system has subsequently been used to investigate differences in pathogenicity of JEV derived from insect and mammalian hosts. Preliminary results suggested both categories of virus were able to replicate to high titers in two porcine cell types. Variability was observed with respect to cytopathic changes, cell viability, and JEV antigen localization. Finally, the virus-vector-host interactions and ecological factors important for JEV transmission and spread, as well as future directions, challenges, and implications of a JEV incursion were reviewed in a collaboration with veterinary epidemiologists at Kansas State University.
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