We elucidated the molecular epidemiology and evolution of Japanese encephalitis virus (JEV) strains isolated from 1949 to 2009 in China in this study. Three genotypes (I, III, V) were confirmed to be co-circulating in China in both high- and low-prevalence areas. Genotype III consisted of two clades (mainland clade and Taiwan clade). Compared to the mainland clade, genotype I and the Taiwan clade were newly introduced and evolved more rapidly. We also demonstrated that JEV strains in China, especially those in the mainland clade, were not only under purifying selection, but also probably under positive selection (aa 227 and 408 in the envelope protein).

Japanese encephalitis (JE) has been found to be endemic in Bali, Indonesia. A case-control study was conducted to identify factors associated with JE infection. All 94 serologically confirmed JE cases (cases) and 163 cases of encephalitis or aseptic meningitis without JE (controls) identified in Bali during 2001–2004 were included in the study. Potential risk factors were surveyed at hospital admission. Univariate analyses revealed the following factors to be associated with JE: older age, referral from sub-district health centre or private hospital, playing outdoors after dinner, use of mosquito repellent or spraying, proximity of the residence to rice fields, and pig ownership by the family or next-door neighbours. Multivariate analysis identified proximity to rice fields (OR 2·93, 95% CI 1·57–5·45), pig ownership (OR 2·24, 95% CI 1·17–4·26), and older age (OR 1·21, 95% CI 1·09–1·33) as being independently associated with the risk of JE. Because rice cultivation and pig rearing are essential to the economy of Bali, JE immunization is the best intervention for prevention of JE in Bali.

Sequences of 191 flavivirus RNAs belonging to four sero-groups were used to predict the secondary structure of the 3′ noncoding region (3′ NCR) directly upstream of the conserved terminal hairpin. In mosquito-borne flavivirus RNAs (n = 164) a characteristic structure element was identified that includes a phylogenetically well-supported pseudoknot. This element is repeated in the dengue and Japanese encephalitis RNAs and centers around the conserved sequences CS2 and RCS2. In yellow fever virus RNAs that contain one CS2 motif, only one copy of this pseudoknotted structure was found. The conserved pseudoknotted element is absent from the 3′ NCR of tick-borne virus RNAs, which altogether adopt a secondary structure that is very different from that of mosquito-borne virus RNAs. The strong conservation of the pseudoknot in mosquito-borne flavivirus RNAs implies a stronger relationship between these viruses than concluded from previous secondary structure analyses. The role of the (tandem) pseudoknots in flavivirus replication is discussed.