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Research Project: Japanese Encephalitis Virus Prevention and Mitigation Strategies

Location: Foreign Arthropod Borne Animal Disease Research

2019 Annual Report

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.

Progress Report
Progress was made on all three sub-objectives which address Component 1 (Biodefense) of the 2016-2021 Animal Health National Program (NP 103) Action Plan. [1A] The attenuated BSL-2 strain of JEV, SA 14-14-2, is known to infect a mosquito cell line derived from Aedes albopictus. While Aedes mosquitoes have been shown to be infected with JEV, Culex species are the prevalent vectors of JEV. Replication kinetic studies were completed and demonstrated infection of SA 14-14 in two Culex cell lines derived from species found in the United States. These data were the first to demonstrate that Culex cell lines were susceptible to infection with the SA 14-14-2 strain. These studies lay the foundation for utilizing these cells lines to examine virus-vector interactions important for pathogenesis and maintenance in North American Culex species mosquitoes. Additionally, this information may promote the use of SA 14-14-2 as a BSL-2 surrogate for JEV studies. Surrogates are important for understanding individual steps crucial for virus replication in various vector and host systems when containment laboratories are not available. [1B] Initial dose-response and replication kinetic studies were performed in porcine cell lines infected with JEV SA 14-14-2. The results indicate that: 1) SA 14-14-2 can replicate in cells originating from multiple porcine tissues, including immune cells, and 2) the amount of infectious virus produced can vary by orders of magnitude between cell types. Notably, our studies with cell lines developed by ARS scientists in Clay Center, Nebraska, suggest that porcine innate immune cells are permissive to JEV infection and permit virus replication at high titers. Together, these results inform our understanding of pathogenesis in the porcine host by indicating which tissues may be more susceptible to infection. Additionally, establishment of this in vitro infection model system will facilitate molecular and cellular studies of JEV infection such as investigations of cell viability/death and virion maturation. [1C] After demonstrating the growth of JEV in cell lines derived from North American species of Culex is possible, timelines to harvest virus from the cells were determined and initial virus stocks were generated. Optimization is ongoing to increase yields and titers to support upcoming studies comparing infectivity of virus propagated in mosquito cells vs. mammalian cells.

1. Japanese encephalitis virus (JEV) is one of the most important etiologic agents for encephalitis worldwide. The virus is maintained in a cycle between culicine mosquitoes and vertebrate hosts. Work with wild-type JEV is strictly controlled and is limited to a Biosafety level-3 containment environment which is used for pathogens that can cause serious or lethal disease. The vaccine strain of JEV can be studied in lower containment lab environments which are common throughout the United States. The vaccine strain is known to grow in mosquito cells. However, the cell line that is typically used is not representative of the mosquito genus associated with most JEV vectors. The JEV vaccine strain was utilized to infect two cell lines derived from culicine mosquitoes. One cell line was derived from Culex quinquefasciatus which are known to be competent vectors for the virus in areas of Asia. The second cell line was derived from a species in the United States that has been shown to be a competent vector in a lab setting. Replication studies demonstrated that the virus produced peak titers after 2-3 days of infection and that the virus did not cause cytopathic effects to the cells. These data suggest that this system could be used as a surrogate for the more virulent viruses in studies examining the molecular mechanisms of the virus-vector interaction important for replication and maintenance in the culicine mosquitoes.

Review Publications
Oliveira, A.S., Piaggio, J., Cohnstaedt, L.W., McVey, D.S., Cernicchiaro, N. 2018. A quantitative risk assessment (QRA) of the risk of introduction of the Japanese encephalitis virus (JEV) in the United States via infected mosquitoes transported in aircraft and cargo ships. Preventive Veterinary Medicine. 160(18)1-9.
Moon, S.A., Cohnstaedt, L.W., McVey, D.S., Scoglio, C.M. 2019. A spatio-temporal individual-based network framework for West Nile virus in the USA: Spreading pattern of West Nile virus. PLoS Computational Biology. 15(3):E1006875.
Oliveira, A.S., Piaggio, J., Cohnstaedt, L.W., McVey, D.S., Cernicchiaro, N. 2019. Introduction of the Japanese encephalitis virus (JEV) in the United States – a qualitative risk assessment. Transboundary and Emerging Diseases. 66:1558-1574.