A Kunjin (KUN) virus cDNA sequence of 10664 nucleotides was obtained and it encoded a single open reading frame for 3433 amino acids. Partial N-terminal amino acid analyses of KUN virus-specified proteins identified the polyprotein cleavage sites and the definitive gene order. The gene order relative to that proposed for yellow fever (YF) virus is as follows: KUN 5′-C·GP20·E·GP44·P19·P10·P71·(?)·P21·P98-3′ YF 5′-C·prM·E·NS1·ns2a·ns2b·NS3·ns4a·ns4b·NS5-3′. The order of putative signal sequences and stop transfer sequences indicated that KUN NS1, NS2A and NS4B are probably cleaved in the lumen of the endoplasmic reticulum, at a consensus site Val-X-Ala↓ where X is an uncharged residue, and NS2B, NS3 and NS5 are cleaved in the cytosol at the site Lys-Arg↓Gly. Comparisons with the complete amino acid sequences of YF and West Nile (WN) viruses showed that KUN virus shared 93% homology with WN virus, but only 46% homology with YF virus. Comparisons among individual gene products of six flaviviruses showed that E, NS1, NS3 and NS5 tended to be the most highly conserved, and C among the least conserved. Homologous cleavage sites were evident, and six domains in NS5, a total of over 170 residues, shared at least 85% homology. Comparisons with the KUN C to NS2B sequence defined a gradient of relationships of all gene products in decreasing order WN > Murray Valley > Japanese encephalitis > St Louis encephalitis viruses within this closely related serological complex. A non-coding 5′ sequence (75 nucleotides) of KUN virus shared 95% homology with WN virus and a shorter imperfect match with Murray Valley encephalitis virus (15 of 18 nucleotides). The KUN non-coding 3′ sequence of 290 nucleotides contained several short and imperfectly matched sequences, and shared 87% homology over the distal region of 191 nucleotides with the corresponding region of WN virus RNA.


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