As it was clear that the JV 59GS did not posses any IRES-like functions it was necessary to determine the location of translation initiation within ORF1. This was predicted be the ATG encoding methionine at nucleotide position 22, as it is situated in a favourable context for translation initiation [7] . To investigate this multiple translation termination codons (polySTOP) were inserted into the JV genome within the 3B-encoding region, downstream of the 59GS, to halt translation at a defined point. In vitro transcription and translation of this construct would, in theory, yield a product whose size would relate to the initiation codon used within the 59GS (Figure 3 ). To address the unlikely event of translation read-through or re-initiation downstream of the polySTOP, which would result in subsequent translation of the 3C protease and cleavage of the truncated ORF1 polyprotein, a mutation was made within the active site encoding region of the 3C protease within JV ORF1, to prevent any viral mediated cleavage of ORF1 translation products (JV 3C mut /polySTOP). A point mutation of the critical cysteine residue within the highly conserved GDCG motif to a glycine residue was performed, and this approach has been described for the successful inactivation of other norovirus' 3C activity [28] . In vitro transcription and translation analysis was performed on JV wild type ( Figure 4 within the 3C region of JV successfully inactivated the 3C protease, thus a large, .200kDa uncleaved polyprotein is yielded following TNTH. The major product generated by JV 3C mut / polySTOP was calculated to be 103kDa in size. Based on computer predictions this is in agreement with the initiation of translation occurring at nucleotide 22, which demonstrates that the JV N-term protein is translated in full in vitro. At this time it is not possible to determine whether translation of intracellular VPgbound viral RNA initiates at nucleotide 22, although it is likely given the favourable context in which the initiation codon is situated.