Investigation into an annonymous, unclaimed Japanese encephalitis virus strain and, the biosafety and biosecurity lessons learnt.

Abstract

Background: Japanese encephalitis (JE) is caused by the mosquito–borne Japanese encephalitis virus (JEV). The transmission cycle of the virus naturally takes place between wild birds and Culex tritaeniorhynchus mosquitoes, with pigs often serving as important natural host due to the high and prolonged viraemia. In year 2009, while searching for specimens in a freezer located in an unsecured room, we stumbled upon a vial labelled as “JEV” with no record of its origin. This vial was treated as a risk group 3 organism and immediately sent to the biocontainment level 3 facility, according to the standard operating procedures, for further investigation. Methods and materials: As a result of the vial label and to test the viability of the organism, the content was inoculated into Vero cell cultures and viewed daily for signs of cytopathic effect (CPE). A novel real time PCR specific for the NS3 region of JEV was designed to confirm the identity of the organism. Additionally, biosafety and biosecurity protocols were assessed and reviewed to address the storage of infectious materials in the laboratory. Results: Cells began to exhibit CPE at day 4 post-inoculation. Melting curve analysis revealed that the real time PCR was specific and sequencing of the purified PCR product confirmed the organism as JEV (strain MY8662). Phylogenetic analysis showed that MY8662 and the Nakayama strain were genetically very similar, evident by the high nucleotide (99.68%) and deduced amino acid (99.88%) sequence similarities.The root cause of the unsecured vial was obscure inventory that failed to record specimens that could have been stored for decades. To this end, an access controlled room with ventilation was constructed to house all freezers storing infectious materials, coupled with an inventory system with barcodes on every storage vial. Additionally, inventory accountability was instituted for the tracking and transportation of infectious materials within and outside of the institution. Conclusion: This unanticipated discovery of an unknown vial has precipitated the laboratory management to take significant measures to address the potential impact of the loss or intentional misuse of these agents. It is therefore, crucial to establish a sustainable culture of safety that involves every stakeholder in the laboratory.