Bacillus anthracis Virulent Plasmid pX02 Genes Found in Large Plasmids of Two Other Bacillus Species

In order to cause the disease anthrax, Bacillus anthracis requires two plasmids, pX01 and pX02, which carry toxin and capsule genes, respectively, that are used as genetic targets in the laboratory detection of the bacterium. Clinical, forensic, and environmental samples that test positive by PCR protocols established by the Centers for Disease Control and Prevention for B. anthracis are considered to be potentially B. anthracis until confirmed by culture and a secondary battery of tests. We report the presence of 10 genes (acpA, capA, capB, capC, capR, capD, IS1627, ORF 48, ORF 61, and repA) and the sequence for the capsule promoter normally found on pX02 in Bacillus circulans and a Bacillus species closely related to Bacillus luciferensis. Tests revealed these sequences to be present on a large plasmid in each isolate. The 11 sequences consistently matched to B. anthracis plasmid pX02, GenBank accession numbers AF188935.1, AE011191.1, and AE017335.3. The percent nucleotide identities for capD and the capsule promoter were 99.9% and 99.7%, respectively, and for the remaining nine genes, the nucleotide identity was 100% for both isolates. The presence of these genes, which are usually associated with the pX02 plasmid, in two soil Bacillus species unrelated to B. anthracis alerts us to the necessity of identifying additional sequences that will signal the presence of B. anthracis in clinical, forensic, and environmental samples

Significant residue diversity between CO clades.

<p>Significant residue diversity between CO clades.</p

Regions of sampling countries^a.

<p>Regions of sampling countries<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006670#t001fn001" target="_blank">^a</a>.</p

Phylogeographic clustering of Caribbean/Americas epidemic CHIKV strains.

<p>The Bayesian maximum clade credibility (MCC) tree of CHIKV Asian genotype was annotated to reflect the geographic origin of samples collected in the Americas. The CO clade was split into three parts, magnified, and indicated by gray shading: subclade CO1 (right side of the tree), subclade CO2 (left middle panel), and non-CO1/CO2 strains (left lower panel). Sequences from different regions are colored in branches as described in the key. The original countries of infections (either indigenous or travel-related) are depicted with colored ovals next to the complete strain names and described in the key as well.</p

Phylodynamics of CHIKV Asian genotype epidemics.

<p>Time-scaled maximum clade credibility (MCC) tree of the CHIKV Asian genotype was inferred by Bayesian analysis. Sequences from different regions are colored as described in the key. The Caribbean outbreak (CO) clade is supported by Bayesian posterior probabilities (BPP) as 1 in BMCMC analysis and bootstrap values 96% in the maximum likelihood phylogeny. Subclades CO1 and CO2, supported by BPP over 0.98, are also marked in the tree. A small group of Nicaraguan sequences that are relatively distinct from other Nicaraguan sequences are highlighted by a red hash symbol (#). The major ancestral geographic states (state probabilities over 0.5) at the backbone of phylogenetic tree are colored by region and labeled in the tree. The nodes with state probabilities greater than 0.9 are marked by asterisks. The mean and 95% highest posterior density (HPD) of the most recent common ancestor (tMRCA) of the entire Asian genotype and tMRCAs of virus movement between regions are also indicated. The scale bar represents chronological time (in years).</p

Maximum likelihood tree of CHIKV Asian genotype.

<p>The major nodes with bootstrap values above 70% are indicated. The CO clade, supported by bootstrap value 96.6%, is colored in red. Amino acid substitutions that contribute to clade diversity are mapped to the branches of the tree. CO-clade-specific amino acid substitutions are marked in red. Monophyletic groups within CO clade and associated amino acid changes are marked in blue. The scale bar represents genetic distance.</p

Differing epidemiological dynamics of Chikungunya virus in the Americas during the 2014-2015 epidemic

<div><p>Chikungunya virus (CHIKV) has been detected sporadically since the 1950s and includes three distinct co-circulating genotypes. In late 2013, the Asian genotype of CHIKV was responsible for the Caribbean outbreak (CO) that rapidly became an epidemic throughout the Americas. There is a limited understanding of the molecular evolution of CHIKV in the Americas during this epidemic. We sequenced 185 complete CHIKV genomes collected mainly from Nicaragua in Central America and Florida in the United States during the 2014–2015 Caribbean/Americas epidemic. Our comprehensive phylogenetic analyses estimated the epidemic history of the Asian genotype and the recent Caribbean outbreak (CO) clade, revealed considerable genetic diversity within the CO clade, and described different epidemiological dynamics of CHIKV in the Americas. Specifically, we identified multiple introductions in both Nicaragua and Florida, with rapid local spread of viruses in Nicaragua but limited autochthonous transmission in Florida in the US. Our phylogenetic analysis also showed phylogeographic clustering of the CO clade. In addition, we identified the significant amino acid substitutions that were observed across the entire Asian genotype during its evolution and examined amino acid changes that were specific to the CO clade. Deep sequencing analysis identified specific minor variants present in clinical specimens below-consensus levels. Finally, we investigated the association between viral phylogeny and geographic/clinical metadata in Nicaragua. To date, this study represents the largest single collection of CHIKV complete genomes during the Caribbean/Americas epidemic and significantly expands our understanding of the emergence and evolution of CHIKV CO clade in the Americas.</p></div

Maximum likelihood phylogenetic tree of complete coding nucleotide sequences of CHIKV.

<p>Well-supported nodes by bootstrap values over 70% are marked in the tree. Three genotypes (West African, ECSA, and Asian) and clades (Indian Ocean and Caribbean outbreaks) are described in the trees. New sequences sampled from the Americas are colored in red. The phylogenetic tree is midpoint-rooted, and the scale bar represents the number of nucleotide substitutions per site. Arrows indicate known recent travel of the human hosts to Africa or Asia in the ECSA genotype.</p