The genome of hepatitis C virus (HCV) contains cis-acting replication elements (CREs) comprised of RNA stem-loop structures located in both the 5 and 3 noncoding regions (5 and 3 NCRs) and in the NS5B coding sequence. Through the application of several algorithmically independent bioinformatic methods to detect phylogenetically conserved, thermodynamically favored RNA secondary structures, we demonstrate a longrange interaction between sequences in the previously described CRE (5BSL3.2, now SL9266) with a previously predicted unpaired sequence located 3 to SL9033, approximately 200 nucleotides upstream. Extensive reverse genetic analysis both supports this prediction and demonstrates a functional requirement in genome replication. By mutagenesis of the Con-1 replicon, we show that disruption of this alternative pairing inhibited replication, a phenotype that could be restored to wild-type levels through the introduction of compensating mutations in the upstream region. Substitution of the CRE with the analogous region of different genotypes of HCV produced replicons with phenotypes consistent with the hypothesis that both local and long-range interactions are critical for a fundamental aspect of genome replication. This report further extends the known interactions of the SL9266 CRE, which has also been shown to form a "kissing loop" interaction with the 3 NCR (P. Friebe, J. Boudet, J. P. Simorre, and R. Bartenschlager, J. Virol. 79:380-392, 2005), and suggests that cooperative long-range binding with both 5 and 3 sequences stabilizes the CRE at the core of a complex pseudoknot. Alternatively, if the long-range interactions were mutually exclusive, the SL9266 CRE may function as a molecular switch controlling a critical aspect of HCV genome replication.Hepatitis C virus (HCV), a flavivirus in the genus Hepacivirus, possesses a positive (mRNA)-sense genome of approximately 9.6 kb encoding a single polyprotein. This polyprotein is cleaved co-and posttranslationally to generate proteins that form the enveloped virus particle and those that replicate the genome. Polyprotein translation is initiated within a highly structured internal ribosome entry site (IRES) occupying much of the 5Ј noncoding region (5Ј NCR). The 5Ј NCR also contains sequences required for genome replication (9,19,26), and like functionally analogous regions in the 3Ј NCR, these form defined stem-loop structures that operate in cis and are known or suspected to recruit cellular or viral proteins (5, 10, 30). In addition to these cis-acting replication elements (CREs) in the noncoding extremes of the genome, there is evidence that additional RNA structures exist within the coding regions. The latter structure is of two types, phylogenetically conserved well-defined structures occupying the 5Ј and 3Ј regions of the sense strand of the coding region of HCV (23,31,32,36) and a less well-characterized but much more extensive set of RNA secondary structures, collectively designated genome-scale ordered RNA structure (GORS), that spans the entire coding ...

K13965, an uncharacterized virus, was isolated in 1993 from Anopheles annulipes mosquitoes collected in the Kimberley region of northern Western Australia. Here, we report its genomic sequence, identify it as a rhabdovirus, and characterize its phylogenetic relationships. The genome comprises a P′ (C) and SH protein similar to the recently characterized Tupaia and Durham viruses, and shows overlap between G and L genes. Comparison of K13965 genome sequence to other rhabdoviruses identified K13965 as a strain of the unclassified Australian Oak Vale rhabdovirus, whose complete genome sequence we also determined. Phylogenetic analysis of N and L sequences indicated genetic relationship to a recently proposed Sandjima virus clade, although the Oak Vale virus sequences form a branch separate from the African members of that group.

BackgroundRecent increased activity of the mosquito-borne Murray Valley encephalitis virus (MVEV) in Australia has renewed concerns regarding its potential to spread and cause disease.Methodology/Principal FindingsTo better understand the genetic relationships between earlier and more recent circulating strains, patterns of virus movement, as well as the molecular basis of MVEV evolution, complete pre-membrane (prM) and Envelope (Env) genes were sequenced from sixty-six MVEV strains from different regions of the Australasian region, isolated over a sixty year period (1951–2011). Phylogenetic analyses indicated that, of the four recognized genotypes, only G1 and G2 are contemporary. G1 viruses were dominant over the sampling period and found across the known geographic range of MVEV. Two distinct sub-lineages of G1 were observed (1A and 1B). Although G1B strains have been isolated from across mainland Australia, Australian G1A strains have not been detected outside northwest Australia. Similarly, G2 is comprised of only Western Australian isolates from mosquitoes, suggesting G1B and G2 viruses have geographic or ecological restrictions. No evidence of recombination was found and a single amino acid substitution in the Env protein (S332G) was found to be under positive selection, while several others were found to be under directional evolution. Evolutionary analyses indicated that extant genotypes of MVEV began to diverge from a common ancestor approximately 200 years ago. G2 was the first genotype to diverge, followed by G3 and G4, and finally G1, from which subtypes G1A and G1B diverged between 1964 and 1994.Conclusions/SignificanceThe results of this study provides new insights into the genetic diversity and evolution of MVEV. The demonstration of co-circulation of all contemporary genetic lineages of MVEV in northwestern Australia, supports the contention that this region is the enzootic focus for this virus.

The Kokobera virus group comprises mosquito-borne flaviviruses that cluster together phylogenetically. These viruses are unique to Australia and Papua New Guinea, and have been associated with a mild polyarticular disease in humans. Recent isolation of genetically diverse viruses within this group has prompted analysis of their genetic and phenotypic relationships. Phylogenetic analysis based on complete ORF, the envelope gene or the NS5/3′ untranslated region supported the separation of the group into distinct species: Kokobera virus (KOKV), Stratford virus, New Mapoon virus, MK7979 and TS5273. Virulence studies in 3-week-old mice also provided the first evidence that a member of the KOKV group (MK7979) was neuroinvasive after intraperitoneal inoculation. In this context, our recent detection of KOKV group-specific antibodies in horses in the field suggests that these viruses should be considered in the epidemiology of flavivirus encephalitis in Australia.