Metadata-driven comparative analysis tool for sequences (meta-CATS): an automated process for identifying significant sequence variations that correlate with virus attributes.

The Virus Pathogen Resource (ViPR; www.viprbrc.org) and Influenza Research Database (IRD; www.fludb.org) have developed a metadata-driven Comparative Analysis Tool for Sequences (meta-CATS), which performs statistical comparative analyses of nucleotide and amino acid sequence data to identify correlations between sequence variations and virus attributes (metadata). Meta-CATS guides users through: selecting a set of nucleotide or protein sequences; dividing them into multiple groups based on any associated metadata attribute (e.g. isolation location, host species); performing a statistical test at each aligned position; and identifying all residues that significantly differ between the groups. As proofs of concept, we have used meta-CATS to identify sequence biomarkers associated with dengue viruses isolated from different hemispheres, and to identify variations in the NS1 protein that are unique to each of the 4 dengue serotypes. Meta-CATS is made freely available to virology researchers to identify genotype-phenotype correlations for development of improved vaccines, diagnostics, and therapeutics.

A phylogenetic analysis using full-length viral genomes of South American dengue serotype 3 in consecutive Venezuelan outbreaks reveals a novel NS5 mutation.

Dengue virus currently causes 50-100 million infections annually. Comprehensive knowledge about the evolution of Dengue in response to selection pressure is currently unavailable, but would greatly enhance vaccine design efforts. In the current study, we sequenced 187 new dengue virus serotype 3 (DENV-3) genotype III whole genomes isolated from Asia and the Americas. We analyzed them together with previously-sequenced isolates to gain a more detailed understanding of the evolutionary adaptations existing in this prevalent American serotype. In order to analyze the phylogenetic dynamics of DENV-3 during outbreak periods; we incorporated datasets of 48 and 11 sequences spanning two major outbreaks in Venezuela during 2001 and 2007-2008, respectively. Our phylogenetic analysis of newly sequenced viruses shows that subsets of genomes cluster primarily by geographic location, and secondarily by time of virus isolation. DENV-3 genotype III sequences from Asia are significantly divergent from those from the Americas due to their geographical separation and subsequent speciation. We measured amino acid variation for the E protein by calculating the Shannon entropy at each position between Asian and American genomes. We found a cluster of seven amino acid substitutions having high variability within E protein domain III, which has previously been implicated in serotype-specific neutralization escape mutants. No novel mutations were found in the E protein of sequences isolated during either Venezuelan outbreak. Shannon entropy analysis of the NS5 polymerase mature protein revealed that a G374E mutation, in a region that contributes to interferon resistance in other flaviviruses by interfering with JAK-STAT signaling was present in both the Asian and American sequences from the 2007-2008 Venezuelan outbreak, but was absent in the sequences from the 2001 Venezuelan outbreak. In addition to E, several NS5 amino acid changes were unique to the 2007-2008 epidemic in Venezuela and may give additional insight into the adaptive response of DENV-3 at the population level.

Evidence for separation of HCV subtype 1a into two distinct clades.

The nucleotide sequence diversity present among hepatitis C virus (HCV) isolates allows rapid adjustment to exterior forces including host immunity and drug therapy. This viral response reflects a combination of a high rate of replication together with an error-prone RNA-dependent RNA polymerase, providing for the selection and proliferation of the viruses with the highest fitness. We examined HCV subtype 1a whole-genome sequences to identify positions contributing to genotypic and phenotypic diversity. Phylogenetic tree reconstructions showed two distinct clades existing within the 1a subtype with each clade having a star-like tree topology and lacking definite correlation between time or place of isolation and phylogeny. Identification of significant phylogenetically informative sites at the nucleotide level revealed positions not only contributing to clade differentiation, but which are located at or proximal to codons associated with resistance to protease inhibitors (NS3 Q41) or polymerase inhibitors (NS5B S368). Synonymous/nonsynonymous substitution mutation analyses revealed that the majority of nucleotide mutations yielded synonymous amino acids, indicating the presence of purifying selection pressure across the polyprotein with pockets of positive selection also being detected. Despite evidence for divergence at several loci, certain 1a characteristics were preserved including the length of the alternative reading frame/F protein (ARF/F) gene, and a subtype 1a-specific phosphorylation site in NS5A (S349). Our analysis suggests that there may be strain-specific differences in the development of antiviral resistance to viruses infecting patients who are dependent on the genetic variation separating these two clades.