Diagnostic and phylogenetic perspectives of the 2023 Murray Valley encephalitis virus outbreak in Australia: an observational study.
BACKGROUND: An outbreak of Murray Valley encephalitis virus (MVEV), the largest since 1974, was observed in Australia between Jan 1 and July 31, 2023. This study aims to characterise the utility of diagnostic platforms, testing algorithms, and genomic characteristics of MVEV to facilitate a comprehensive framework for MVEV testing and surveillance in the outbreak setting. METHODS: In this observational study, we assessed flavivirus diagnostics for all patients with suspected Murray Valley encephalitis in Australia from Jan 1 to July 31, 2023. We included all patients with confirmed Murray Valley encephalitis, probable Murray Valley encephalitis, or acute unspecified flavivirus infection using the Communicable Diseases Network Australia case definition. Cases were excluded if an alternative diagnosis was identified. We collected blood, serum, cerebrospinal fluid, brain tissue, urine, or a combination of these samples, as appropriate and at the discretion of the treating clinician. We conducted multimodal diagnostic testing, which included flavivirus-specific serological and nucleic acid amplification testing. Metagenomic next-generation sequencing, including next-generation deep sequencing, target-enrichment, and targeted amplification, was conducted on human and representative mosquito-derived samples obtained from established mosquito population surveillance programmes for phylogenetic analysis. FINDINGS: 27 patients with encephalitis were assessed for MVEV between Jan 1, 2023, and July 31, 2023, 23 (85%) of whom fulfilled national case definitions for confirmed Murray Valley encephalitis. Patient ages ranged from 6 weeks to 83 years (median 62·0 years [IQR 31·0-67·5]) and patients were mostly male (21 [78%] male patients and six [22%] female patients). Incidence varied widely by geographical region and was highest in the Northern Territory (32·0 per 1 000 000 population). Diagnostic specimen collection generally occurred promptly (median 6·0 days [IQR 4·0-14·5] from symptom onset to diagnostic specimen collection). In seven patients, case assignation relied on convalescent serum samples to assess for seroconversion or an appropriate rise in antibody titre (to four times the initial value or greater), or both. MVEV-specific IgM was detectable in serum samples of 17 (81%) of 21 patients tested by day 7 and MVEV IgG or total antibody (TAb) were detected in 18 (100%) of 18 patients tested by day 30. MVEV-specific IgM (or TAb) and MVEV RNA were detected in cerebrospinal fluid collected within 14 days of symptom onset in nine (39%) of 23 patients and seven (28%) of 25 patients, respectively. Phylogenetic analysis revealed two circulating MVEV genotypes, G1A and G2, in mosquitoes and humans in 2023. In southeast Australia, only G1A was detected and probably introduced from enzootic foci in northern Australia. INTERPRETATION: This study provides a comprehensive overview of the diagnostic workflows and phylogenetic evaluations used during the 2023 MVEV outbreak in Australia, emphasising the importance of a multimodal approach for accurate and timely confirmation of flavivirus infection. Further One Health surveillance for MVEV and other zoonotic flaviviruses is key, given potential expanded ecological niches in the context of episodic climatic events. FUNDING: None.