Location: Foreign Arthropod Borne Animal Disease Research
2020 Annual Report
Objectives
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.
Approach
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
Research continued on Objective 1, our sole objective. We continued to make progress towards understanding Japanese encephalitis virus (JEV) maintenance in relevant mosquito vectors using a biosafety level 2 (BSL-2) cell model system that we developed in fiscal year 2019. JEV is known to grow in mosquito cells; however, the cell line typically used is not representative of the predominate vectors for the virus. To determine the ability of JEV to replicate in a more relevant cell line, the vaccine strain of JEV was used to infect two cell lines derived from the primary group of mosquitoes. Replication studies demonstrated that this strain of JEV grows to high titers in one of the cell lines. This cell line, which was recently developed by ARS scientists at Manhattan, Kansas, and represents a North American mosquito species, provided sufficient concentrations of virus for use in future studies. The virus infection did not cause any noticeable detrimental effects to the cell line, depicting a natural infection in the mosquito. This is different than what occurs in the more commonly used and less relevant mosquito cell line, in which virus does cause detrimental effects to the cells after 5 days of infection. These data indicate that the new cell line is relevant for stock production and for use in studies examining the molecular mechanisms of the virus-vector interaction important for replication and maintenance in mosquito vectors. Progress likewise continued towards characterizing how JEV develops in swine, the main amplifying animal host in the JEV transmission cycle. A BSL-2 surrogate system that we developed in fiscal year 2019 was further optimized and used to study cell lines originating from pig tissues that were known or hypothesized to be susceptible to infection with JEV. This susceptibly to virus infection was studied by measuring cell death and observing cytopathic effects (CPE) in the infected cells. The amount of infectious virus maintained within the cells versus released was also measured to determine the ability of the cells to support a productive viral infection. Several of the cell lines produced virus at levels comparable to a common model cell line, BHK, which is known to be highly susceptible to JEV infection and is commonly used to grow high titer virus stocks. Notably, unlike BHK cells, some of the pig cell lines experienced no cell death or CPE while producing large amounts of virus. Based on these findings, we have identified cell types to take forward into future studies and to use for the growth of virus stocks. Progress was made towards our research with wild-type JEV. Work with wild-type JEV is strictly controlled and limited to biosafety level-3 (BSL-3) containment labs. Our studies with the attenuated vaccine strain have been instrumental in establishing and optimizing methods which are now ready to transition for use in the higher-level BSL-3 lab. We successfully completed the lengthy process to obtain our foundational wild-type viruses after it was determined a new source was required. We received the viruses and have prepared initial virus stocks. We collaborated with veterinary epidemiologists at Kansas State University to review 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. This collaboration resulted in a publication discussing perspectives regarding the risk of introduction of the JEV in the United States.
Accomplishments
1. Analysis of U.S. vulnerabilities to introduction of Japanese encephalitis virus. Japanese encephalitis virus (JEV) is a mosquito-transmitted virus that can infect multiple animal species and cause severe disease and death in humans. JEV is endemic in Asia and the Pacific region and is not currently present in the United States (U.S.). However, JEV has demonstrated the ability to spread to new geographic regions and previous studies determined the U.S. is at high risk for JEV introduction. ARS researchers in Manhattan, Kansas collaborated with researchers at Kansas State University to better understand challenges and consequences associated with potential JEV introduction into the US. Their analysis highlighted several areas of vulnerability including increases in wild animal populations that can transmit the disease, illegal animal importation and movement, decreases in mosquito control in regions with highest risk, and changing patterns of viral strains. Identifying these gaps can help target animal and mosquito monitoring to more rapidly detect an incursion of JEV. This information will also be useful to direct future research, such as studies of the effectiveness of existing vaccines against different strains of JEV. Efforts such as these can reduce the potential consequences of a JEV incursion in the US.
