Molecular phylogenetic and evolutionary analyses of Muar strain of Japanese encephalitis virus reveal it is the missing fifth genotype. 
Japanese encephalitis virus (JEV) is the most important cause of epidemic encephalitis worldwide but its origin is unknown.  Epidemics of encephalitis suggestive of Japanese encephalitis (JE) were described in Japan from the 1870s onwards.  Four genotypes of JEV have been characterised and representatives of each genotype have been fully sequenced.  Based on limited information, a single isolate from Malaysia is thought to represent a putative fifth genotype.  We have determined the complete nucleotide and amino acid sequence of Muar strain and compared it with other fully sequenced JEV genomes.  Muar was the least similar, with nucleotide divergence ranging from 20.2 to 21.2% and amino acid divergence ranging from 8.5 to 9.9%.  Phylogenetic analysis of Muar strain revealed that it does represent a distinct fifth genotype of JEV.  We elucidated Muar signature amino acids in the envelope (E) protein, including E327 Glu on the exposed lateral surface of the putative receptor binding domain which distinguishes Muar strain from the other four genotypes.  Evolutionary analysis of full-length JEV genomes revealed that the mean evolutionary rate is 4.35 × 10(-4) (3.4906 × 10(-4) to 5.303 × 10(-4)) nucleotides substitutions per site per year and suggests JEV originated from its ancestral virus in the mid 1500s in the Indonesia-Malaysia region and evolved there into different genotypes, which then spread across Asia.  No strong evidence for positive selection was found between JEV strains of the five genotypes and the E gene has generally been subjected to strong purifying selection.