Japanese encephalitis virus is a leading cause of neurological
infection in the Asia-Pacific region with no means of detection in
more remote areas. We aimed to test the hypothesis of a Japanese encephalitis
(JE) protein signature in human cerebrospinal fluid (CSF) that could
be harnessed in a rapid diagnostic test (RDT), contribute to understanding
the host response and predict outcome during infection. Liquid chromatography
and tandem mass spectrometry (LC–MS/MS), using extensive offline
fractionation and tandem mass tag labeling (TMT), enabled comparison
of the deep CSF proteome in JE vs other confirmed neurological infections
(non-JE). Verification was performed using data-independent acquisition
(DIA) LC–MS/MS. 5,070 proteins were identified, including 4,805
human proteins and 265 pathogen proteins. Feature selection and predictive
modeling using TMT analysis of 147 patient samples enabled the development
of a nine-protein JE diagnostic signature. This was tested using DIA
analysis of an independent group of 16 patient samples, demonstrating
82% accuracy. Ultimately, validation in a larger group of patients
and different locations could help refine the list to 2–3 proteins
for an RDT. The mass spectrometry proteomics data have been deposited
to the ProteomeXchange Consortium via the PRIDE partner repository
with the dataset identifier PXD034789 and 10.6019/PXD034789.