Discovery of a Japanese Encephalitis Virus Nakayama Substrain Highlights the Dual Lessons of Molecular Characterization and Laboratory Biosafety

Abstract

Japanese encephalitis virus (JEV) remains an important cause of viral encephalitis in Asia, with high mortality and neurological sequelae. During a routine inventory at a Malaysian research institution, an unregistered vial labeled “JEV” was recovered from an unsecured −80 °C freezer. The sample, designated MY8662, was investigated under biosafety level 3 conditions. Inoculation of Vero cells produced cytopathic effects, and real-time RT-PCR confirmed JEV identity. Full-genome sequencing revealed 99.68% nucleotide and 99.88% amino acid similarity to the Nakayama vaccine strain, with 35 nucleotide substitutions resulting in four amino acid changes, including novel substitutions in the envelope and NS4A proteins. Phylogenetic analysis clustered MY8662 closely with Nakayama, suggesting a substrain relationship. Replication kinetics in Vero cells showed exponential viral RNA increase with a doubling time of 2.6 hours, comparable to other JEV strains. The incident also revealed biosafety concerns associated with obscure inventories, prompting corrective measures including access-controlled biorepositories and barcode-based inventory systems. This case illustrates the dual significance of forgotten laboratory materials, providing insights into JEV genomic diversity while underscoring the importance of sustainable biosafety and biosecurity practices. Strengthened institutional oversight and risk-based management are essential to prevent accidental exposures or loss of infectious agents.