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Research Project: Japanese Encephalitis Virus Prevention and Mitigation Strategies

Location: Foreign Arthropod Borne Animal Disease Research

2021 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 the single Objective (1) of this project. To better understand Japanese encephalitis virus (JEV) maintenance in relevant mosquito vectors, a new wildtype Asian JEV strain was acquired and used to infect two species of North American Culex mosquitoes in a Biosafety Level-3 (BSL-3) containment 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 affect 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 also made towards understanding JEV maintenance in porcine hosts. A Biosafety Level-2 containment lab in vitro porcine cell system was established and used to investigate differences in pathogenicity of JEV derived from insect and mammalian hosts. Preliminary results suggest 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.

1. Susceptibility of North American mosquitoes to an Asian strain of Japanese encephalitis virus (JEV). Japanese encephalitis virus (JEV) is a mosquito-borne virus that can cause significant disease in animals and humans. There are several genetic variations of JEV. While JEV is currently not found in the United States, some variations have encroached into new countries, making the introduction of JEV into the United States a major concern due to the presence of animal and human populations with no immunity against the disease. To understand the risk of JEV introduction into the U.S., the ability of the different variations to infect North American mosquitoes and host must be examined. ARS scientists in Manhattan, Kansas, studied the ability of an Asian strain of JEV to infect two species of North American mosquitoes. This JEV strain infected both mosquito species and the virus disseminated to the salivary glands suggesting the mosquitoes can transmit the virus to animal hosts. This is the first study to examine this important virus strain in these mosquitoes. These findings will help strengthen JEV risk assessments and have the potential to shape future strategies for surveillance, outbreak response, vaccine development, and vector control.

2. Establishment of an in vitro system to study swine infections with Japanese encephalitis virus (JEV). Japanese encephalitis is a mosquito-transmitted disease which occurs primarily in Asia. It can lead to severe disease and death in humans and reproductive losses in swine. Pigs play an important role in the transmission of Japanese encephalitis virus (JEV) because they can amplify the virus to high levels when they are infected. However, the way that JEV behaves in the pig’s body during infection is not well understood. ARS scientists in Manhattan, Kansas, collaborated with ARS scientists in Clay Center, Nebraska, and Kansas State University scientists to identify a system for studying the biology of JEV in pig cells in vitro. They found that a vaccine strain of JEV could successfully infect, and replicate in, established cell lines derived from four different pig tissues. These findings show that these porcine cell lines have the potential to be useful research tools for investigating JEV infection of pigs which are a natural host species. They may also serve as screening tools for identifying new drugs and other therapeutic treatments.

Review Publications
Ferdousi, T., Cohnstaedt, L.W., McVey, D.S., Scoglio, C.M. 2019. Understanding the survival of Zika virus in a vector interconnected sexual contact network. Scientific Reports. 9:7253.
Wang, L., Madera, R., Li, Y., McVey, D.S., Drolet, B.S., Shi, J. 2020. Recent advances in diagnosis of classical swine fever and future perspectives. Pathogens. 9(8):658.