Enhanced Neutralization of Japanese Encephalitis Virus Using an Engineered VL Double-Domain

Japanese encephalitis virus (JEV) is an emerging virus responsible for thousands of deaths in Asia; however, an effective treatment has not yet been discovered. JEV envelope (E) proteins play important roles in viral survival and infection. In this study, we isolated scFv- and VL-targeting E proteins using phage display biopanning. The isolated VL was engineered to form VL double domains to increase its neutralization activity. Several candidates showed binding abilities, whereas only the DE2 VL double-domain treatment showed neutralizing activity against JEV. DE2 was also highly specific to JEV and did not bind to other flaviviruses. Immunocytochemistry results showed that DE2 was co-localized with the endosome 4 h post-infection. Thus, the mechanism of DE2 neutralization involves blocking the E protein from fusing with the endosomal membrane, resulting in the unrelease of the viral genome into the cytoplasm. Docking analysis showed that DE2 was bound to three domains, especially DIII, of the JEV E protein. In contrast, other scFv that were unable to bind to DIII showed no or low neutralizing activity. DE2 was also predicted to neutralize all the JEV genotypes. In conclusion, a combined in vitro and in silico study showed the high potential of DE2 to neutralize JEV infections, which may be applied in future therapeutic systems. ImportanceJapanese encephalitis virus (JEV) is a serious mosquito-borne disease that causes brain infections and deaths across Asia. However, there is still no specific treatment once infection occurs. In this study, we developed a new type of antibody fragment, called a VL double-domain, that can block the virus. Among several candidates, DE2 showed a strong ability to stop JEV from infecting cells, while others could bind to the virus but did not block infection. Our findings reveal that DE2 works by attaching to a key region of the virus, preventing it from releasing its genetic material. Moreover, DE2 is predicted to work against all known JEV strains, highlighting its potential as a universal treatment option. This research provides an important step toward developing new antiviral therapies for JEV and possibly for other viruses with similar infection mechanisms.