Universal primers that amplify RNA from all three flavivirus subgroups

Background: Species within the Flavivirus genus pose public health problems around the world. Increasing cases of Dengue and Japanese encephalitis virus in Asia, frequent outbreaks of Yellow fever virus in Africa and South America, and the ongoing spread of West Nile virus throughout the Americas, show the geographical burden of flavivirus diseases. Flavivirus infections are often indistinct from and confused with other febrile illnesses. Here we review the specificity of published primers, and describe a new universal primer pair that can detect a wide range of flaviviruses, including viruses from each of the recognised subgroups

Accumulating Variation at Conserved Sites in Potyvirus Genomes Is Driven by Species Discovery and Affects Degenerate Primer Design

Unknown and foreign viruses can be detected using degenerate primers targeted at conserved sites in the known viral gene sequences. Conserved sites are found by comparing sequences and so the usefulness of a set of primers depends crucially on how well the known sequences represent the target group including unknown sequences. Methodology/Principal Findings: We developed a method for assessing the apparent stability of consensus sequences at sites over time using deposition dates from Genbank. We tested the method using 17 conserved sites in potyvirus genomes. The accumulation of knowledge of sequence variants over 20 years caused ‘consensus decay ’ of the sites. Rates of decay were rapid at all sites but varied widely and as a result, the ranking of the most conserved sites changed. The discovery and reporting of sequences from previously unknown and distinct species, rather than from strains of known species, dominated the decay, indicating it was largely a sampling effect related to the progressive discovery of species, and recent virus mutation was probably only a minor contributing factor. Conclusion/Significance: We showed that in the past, the sampling bias has misled the choice of the most conserved target sites for genus specific degenerate primers. The history of sequence discoveries indicates primer designs should be update

The variable codons of H3 influenza A virus haemagglutinin genes

We have analyzed several sets of well-studied haemagglutinin (HA) gene sequences of H3 subtype influenza A viruses to identify codons that are unusually variable, using a simple pairwise sliding window method, DnDscanning. For two of the sets there were results of detailed phylogenetic modeling studies of selection already published. A third set had been the subject of an antigen mapping study, the results of which provide a completely independent benchmark of selected changes in H3 HA genes. Our analyses show that the codons with greatest DnDscan scores (i.e. the most variable) were mostly those reported in the published studies as being positively selected; indeed the DnDscan results matched the antigenic mapping results more closely than did those of the phylogenetic modeling methods. These results suggest that codons under selection can be found even when, as with some sets of virus sequences, a phylogeny is uncertain or cannot be obtained because, for example, the sequences are recombinants, or when selection is not necessarily linked with phylogeny, as in host-switching events. The program DnDscan is available at http://biojanus.anu. edu.au