## 4 Discussion  
Here we present the first known isolation and subsequent full genome sequencing of MVEV from CSF. Historically MVEV diagnosis by isolation and/or PCR has been difficult due to factors such as low viral burden, limited availability of appropriate samples, and infrequency of cases. The last report of multiple isolations from human specimens was from the 1974 outbreak where isolation was attempted on a number of samples, (including 11 throat swabs and 14 CSF) from 24 patients, 22 of which were admitted to our laboratory, then located at Fairfield hospital [7]. In that report, MVEV was only successfully isolated from post-mortem brain tissue, (specifically 6 samples from 3 patients). The systems utilized to cultivate the virus were 11-12 days embryonated eggs, cell lines (Monkey Embryonic Kidney, (MEK) and Vero) and newborn mice. While eggs were the most sensitive method, MVEV was also successfully cultured in MEK cells from two samples (one of which also grew in Vero). More recently, isolation of MVEV from post-mortem brain tissue of a deceased patient from the 2011 outbreak has also been described [8]. Isolation of the MVEV from cell culture of the clinical specimens has not subsequently been reported, perhaps reflecting that techniques of virus isolation have given way to PCR due to its increased speed, sensitivity and specificity, and reduced demand for specific expertise compared to \begin{table}
\begin{tabular}{c c c} \hline \hline
**Amino Acid Change** & **Protein** & **Non-Structural Protein Function** \\ \hline P11S & core protein & \\ A10T & signal peptide & \\ S7G & envelope protein & \\ V68I & envelope protein & \\ L311F & envelope protein & \\ \hline R33K & NS1 protein & \\ V188I & NS1 protein & Early replication [12] \\ D240E & NS1 protein & \\ \hline F1L & NS2A protein & \\ S93T & NS2A protein & \\ I197V & NS2A protein & encapsidation [12] \\ \hline D27E & NS2B protein & \\ N59D & NS2B protein & \\ A67T & NS2B protein & \\ S106G & NS2B protein & \\ I110T & NS2B protein & \\ \hline P131S & NS3 protein & \\ V241I & NS3 protein & \\ G321S & NS3 protein & \\ \hline I54V & NS4A protein & \\ L69F & NS4A protein & NS3 cofactor [12] \\ D98E & NS4A protein & \\ \hline L21F & NS4B protein & \\ L32F & NS4B protein & \\ T69N & NS4B protein & RNA accumulation [12] \\ N76S & NS4B protein & \\ A115V & NS4B protein & \\ \hline R101K & NS5 protein & \\ T116M & NS5 protein & \\ I374T & NS5 protein & \\ K403E & NS5 protein & \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Comparison of VIDRL-MVE to the G2 reference strain MVE-OR156 indicating the 31 predicted non-synonymous amino acid changes. tissue culture. In contrast, this case highlights that cell culture may be more achievable than has been assumed, and supports its use in MVEV genotyping and investigation. The clinical significance of MVEV isolation from a CSF sample with low levels of virus is two-fold. Firstly, it contradicts the perceived superiority (sensitivity) of PCR. We were able to isolate MVEV from CSF yielding a low level Real-Time PCR detection (Ct 41) by inoculating with a large volume. Despite requiring a minimum sample volume of 140-200 \(\upmu\)L for nucleic acid extraction, PCR only allows for an input that is equivalent to approximately 4-10 \(\upmu\)L of that original sample. By comparison, our inoculum volume was 500 \(\upmu\)L, allowing for a higher input of virus particles, thereby potentially increasing the relative sensitivity of the technique when close to the limit of detection. PCR detection and isolation of MVEV from CSF emphasizes its usefulness as a sample for primary diagnosis, with downstream applications for whole genome sequencing. CSF is far preferable to brain tissue, (often acquired post-mortem) and more sensitive than plasma, which must be obtained before the appearance of antibodies clear the viremia [3], consequently, when plasma is no longer useful, CSF may still present as a viable option.