## Materials and Methods
### Nucleic Acid Extraction, Reverse Transcription, PCR Detection, and Sequencing  
CSF from the patient was received in our laboratory in May 2015 from the Royal Darwin Hospital, accompanied by a request for MVEV PCR. Pan-_Flavivirus_ conventional PCR and MVE-specific real-time PCR were performed according to our standard laboratory algorithm for diagnosis. Prior to extraction the specimen was treated with lysis buffer spiked with low copy number bovine diarrheal virus (BVDV) to act as internal control for the nucleic acid extraction, reverse transcription, and PCR amplification. This approach of using a non-human virus to act as an internal control has been previously described by our laboratory [9; 10]. Viral nucleic acid was extracted from 200 \(\upmu\)L patient CSF using the Qiagen QiaCUBE automated extraction robot with Qiagen reagents and consumables (Qiagen, Hilden, Germany) and eluted in 70 \(\upmu\)L AVE elution buffer as per the manufacturer's instructions. 10 \(\upmu\)L of nucleic acid extract underwent reverse transcription using the SensiFast cDNA synthesis kit (BIOLINE, London, UK) as per the manufacturer's instructions.  
The conventional PCR utilizes highly degenerate pan-_Flavivirus_ primers, modified from [PERSON] et al., 2001 which target the NS5 region [11]. 2.5 \(\upmu\)L of cDNA template was added to the master mix containing 0.5 \(\upmu\)M of first or second round primer pool, 1.8 mM Mg Cl\({}_{2}\), 2 \(\upmu\)M dNTPs and 0.3 U of Taq polymerase (Qiagen) to a final volume of 40 \(\upmu\)L. First round PCR cycling conditions consisted of one cycle of 94 \({}^{\circ}\)C for 3 min, followed by 35 cycles of 94 \({}^{\circ}\)C (20 s), 54 \({}^{\circ}\)C (20 s), 72 \({}^{\circ}\)C (30 s) with a final extension of 72 \({}^{\circ}\)C (5 min). Second round PCR cycling conditions consisted of one cycle of 94 \({}^{\circ}\)C for 3 min, followed by 25 cycles of 94 \({}^{\circ}\)C (20 s), 53 \({}^{\circ}\)C (20 s), 72 \({}^{\circ}\)C (30 s) with a final extension of 72 \({}^{\circ}\)C (5 min). Separation of amplified material and molecular weight markers (Bioline Hyperladder 100 bp) was performed by electrophoresis for 30 min at 100 mA on a 2.25% agarose gel pre-stained with ethidium bromide (Sigma-Aldrich, St. Louis, MO, USA) in 1\(\times\) TAE (40 mM tris-acetate [pH 7.6], and 1 mM sodium EDTA). Gels were imaged under UV illumination using a Gel Doc (Biorad, Hercules, CA, USA).  
For real time PCR, 2.5 \(\upmu\)L of cDNA template was added to a final volume of 20 \(\upmu\)L of commercially available fast master-mix (PerfeCTaqPCR FastMix, UNG, Low ROX, QuantaBio, Boston, MA, USA) containing MVE-specific primers (0.9 \(\upmu\)M) and probe (0.2 \(\upmu\)M) (Forward primer 5\({}^{\prime}\)-CAGGCCAGCCGTTAGG, reverse primer 5\({}^{\prime}\)-GGTTCTGAGGCTTTCC, probe 5\({}^{\prime}\)[6 FAM] CCAACCCCGAGAGGA[MGBNFQ]). Runs were performed on an Applied Biosystems (ABI) 7500 Fast Real-time PCR machine using standard cycling conditions of 95 \({}^{\circ}\)C for 20 s followed by 45 cycles of 95 \({}^{\circ}\)C (3 s), and 60 \({}^{\circ}\)C (30 s). The same procedure was followed for the BVDV internal control, (primers and probe sequences available on request).  
A nuclease free water negative, and a virus positive control of low copy number were included for both conventional and Real-time PCR.  
MVEV detection was confirmed by sequencing the second round conventional pan-_Flavivirus_ PCR amplified product. Reaction products were cleaned using the ExoSap-IT kit (Amersham Biosciences, Amersham, England) before sequencing using the ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction Kit. Primers used were the same as per the second round of amplification. Sanger sequencing was performed at Micromon (Monash University), using an ABI 3730 genetic analyser). Murray Valley encephalitis was confirmed by the use of GenBank Basic Local Alignment Tool (BLAST), ([[http://www.ncbi.nlm.nih.gov/Blast](http://www.ncbi.nlm.nih.gov/Blast)]([http://www.ncbi.nlm.nih.gov/Blast](http://www.ncbi.nlm.nih.gov/Blast))).