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The detection of a new and unexpected Japanese encephalitis virus (JEV) outbreak in March 2022 in Australia, where JEV is not endemic, demanded the rapid development of a robust diagnostic framework to facilitate the testing of suspected patients across the state of New South Wales (NSW). This nascent but comprehensive JEV diagnostic service encompassed serological, molecular and metagenomics testing within a centralised reference laboratory. Over the first three months of the outbreak (4 March 2022 to 31 May 2022), 1,061 prospective samples were received from 878 NSW residents for JEV testing. Twelve confirmed cases of Japanese encephalitis (JE) were identified, including ten cases diagnosed by serology alone, one case by metagenomic next generation sequencing and real-time polymerase chain reaction (RT-PCR) of brain tissue and serology, and one case by RT-PCR of cerebrospinal fluid, providing an incidence of JE over this period of 0.15/100,000 persons in NSW. As encephalitis manifests in &lt;1% of cases of JEV infection, the population-wide prevalence of JEV infection is likely to be substantially higher. Close collaboration with referring laboratories and clinicians was pivotal to establishing successful JEV case ascertainment for this new outbreak. Sustained and coordinated animal, human and environmental surveillance within a OneHealth framework is critical to monitor the evolution of the current outbreak, understand its origins and optimise preparedness for future JEV and arbovirus outbreaks.

Emerging Genotype IV Japanese Encephalitis Virus Outbreak in New South Wales, Australia