Japanese encephalitis virus, a mosquito-borne virus, can infect many species including swine and humans. While JEV is not currently in the US, incursion risk remains a concern given the virus’s recent global expansion. The presence of JEV in the US could have devastating outcomes for the swine industry because of its severe disease presentation in neonatal piglets and pregnant sows. In the event of an outbreak, accurate JEV diagnostics will be critical to disease monitoring. Currently, no approved diagnostic tests for JEV antibodies in swine exist in the US. As part of the Swine Health Information Center JEV Research Program, funded in collaboration with the Foundation for Food & Agriculture Research, a recently completed project led by Dr. Katharine Bossart, Integrated Research Associates, LLC, sought to build diagnostic testing capabilities for JEV in the US.
The main goal of the project was to build diagnostic capability for JEV based on diagnostic serological assays used and validated throughout Southeast Asia. JEV is classified as a biosafety level 3 agent, which limits the number of laboratories able to work with the virus and further reduces JEV diagnostic capability. Recognizing this, Dr. Bossart and team evaluated and developed JEV serology assays based on similar tests utilized overseas. These assays use nonhazardous recombinant JEV virus-like particles as viral antigens to broaden access and increase JEV testing capacity.
Read the industry summary for this study, #24-061, here.
JEV has been found throughout Asia and the Western Pacific with many of these countries having active surveillance programs in place for mosquitoes and swine. In countries outside of the region, including the US, JEV surveillance programs have not been implemented and vaccination is only advised in those traveling to countries where JEV is endemic. In 2022, a JEV genotype IV virus caused a widespread and devastating outbreak in eastern Australia in commercial piggeries and in humans. Currently, there are no approved diagnostics for JEV antibodies in humans or animals in the US. Initially, recombinant JEV virus-like particles (VLPs) were being made using a novel technology developed to explore their potential as JEV vaccine candidates. Expanding the use of recombinant JEV VLPs to replace inactivated JEV in diagnostic kits was a logical step towards not just building JEV diagnostic capability but also simplifying kit manufacturing.
To complete this study, sufficient quantities of inactivated JEV viral antigens, JE1, FE1, JN1, JN2 and FN1 monoclonal antibodies (mAbs) and corresponding mAb hybridomas, were imported from Venture Technologies. Stable cell lines expressing JEV VLPs from genotypes III, IV and V were used to produce recombinant JEV VLPs. Although recombinant JEV VLPs were made from genotypes III, IV and V, there were unique properties for each that were most notable for JEV V. Results indicated that JEV VLPs performed well and can replace inactivated JEV in diagnostic kits. Multiple genotypes of JEV can be included as antigens, which represents an improvement in design, and a scalable cGMP-compliant manufacturing process has been developed for recombinant JEV VLPs, which will simplify future regulatory filings and approvals.
Two diagnostic prototype kits containing the new JEV antigens were assembled as part of this trial. One is an ELISA kit for screening numerous serum samples in the laboratory and the second is a dot enzyme immunoassay kit for screening individual, or a few, serum samples on farm sites without advanced technology. Prototype kits are currently being evaluated overseas using approximately 500 swine sera from clinically confirmed cases of JEV and results should be available in late summer 2026.
If a large-scale JEV outbreak occurs in the US, the possibility to utilize vaccination exists. In this scenario, the ability to differentiate vaccinated and infected swine becomes important to biosecurity and control. Therefore, the last objective of the project focused on determining if a recombinant JEV nonstructural protein could be used as an alternate antigen in diagnostic kits as only infected people or infected animals (not vaccinated) make an immune response to nonstructural proteins. A stable cell line expressing the differential antigen was made and extensively characterized using a variety of biochemical and immunological assays. Preliminary data suggest prototype kits will not work with the alternate recombinant nonstructural antigen; however, a different diagnostic serology platform was piloted which could prove useful in an outbreak.
Successful development of new broadly available diagnostic tests for JEV provides the swine industry with the necessary tool to detect recent JEV exposure and/or infection in pigs, aiding biosecurity and control, which will help mitigate the spread and impact of a JEV outbreak in the US. As part of SHIC’s role in surveillance and discovery of emerging diseases, advancing diagnostic capabilities to detect JEV antibodies in swine helps to enhance preparedness for the US swine herd.
Foundation for Food & Agriculture Research
The Foundation for Food & Agriculture Research (FFAR) builds public-private partnerships to fund bold research addressing big food and agriculture challenges. FFAR was established in the 2014 Farm Bill to increase public agriculture research investments, fill knowledge gaps and complement the U.S. Department Agriculture’s research agenda. FFAR’s model matches federal funding from Congress with private funding, delivering a powerful return on taxpayer investment. Through collaboration and partnerships, FFAR advances actionable science benefiting farmers, consumers and the environment.
Swine Health Information Center
The Swine Health Information Center, launched in 2015 with Pork Checkoff funding, protects and enhances the health of the US swine herd by minimizing the impact of emerging disease threats through preparedness, coordinated communications, global disease monitoring, analysis of swine health data, and targeted research investments. As a conduit of information and research, SHIC encourages sharing of its publications and research. Forward, reprint, and quote SHIC material freely. For more information, visit http://www.swinehealth.org or contact Dr. Megan Niederwerder at [email protected] or Dr. Lisa Becton at [email protected].
