Anthrax, caused by the bacterium Bacillus anthracis, is a disease of historical and current importance that is found throughout the world. The basis of its historical transmission is anecdotal and its true global population structure has remained largely cryptic. Seven diverse B. anthracis strains were whole-genome sequenced to identify rare single nucleotide polymorphisms (SNPs), followed by phylogenetic reconstruction of these characters onto an evolutionary model. This analysis identified SNPs that define the major clonal lineages within the species. These SNPs, in concert with 15 variable number tandem repeat (VNTR) markers, were used to subtype a collection of 1,033 B. anthracis isolates from 42 countries to create an extensive genotype data set. These analyses subdivided the isolates into three previously recognized major lineages (A, B, and C), with further subdivision into 12 clonal sub-lineages or sub-groups and, finally, 221 unique MLVA15 genotypes. This rare genomic variation was used to document the evolutionary progression of B. anthracis and to establish global patterns of diversity. Isolates in the A lineage are widely dispersed globally, whereas the B and C lineages occur on more restricted spatial scales. Molecular clock models based upon genome-wide synonymous substitutions indicate there was a massive radiation of the A lineage that occurred in the mid-Holocene (3,064–6,127 ybp). On more recent temporal scales, the global population structure of B. anthracis reflects colonial-era importation of specific genotypes from the Old World into the New World, as well as the repeated industrial importation of diverse genotypes into developed countries via spore-contaminated animal products. These findings indicate humans have played an important role in the evolution of anthrax by increasing the proliferation and dispersal of this now global disease. Finally, the value of global genotypic analysis for investigating bioterrorist-mediated outbreaks of anthrax is demonstrated.

The intracellular bacterium Francisella tularensis is the causative agent of tularemia and poses a serious threat as an agent of bioterrorism. We have developed a highly effective molecular subtyping system from 25 variable-number tandem repeat (VNTR) loci. In our study, multiple-locus VNTR analysis (

Subpopulations A.I and A.II. of Francisella tularensis subsp. tularensis are associated with unique biotic and abiotic factors that maintain disease foci.

Francisella tularensis, the etiological agent of tularemia, is found throughout the Northern hemisphere. After analyzing the F. tularensis genomic sequence for potential variable-number tandem repeats (VNTRs), we developed a multilocus VNTR analysis (MLVA) typing system for this pathogen. Variation was detected at six VNTR loci in a set of 56 isolates from California, Oklahoma, Arizona, and Oregon and the F. tularensis live vaccine strain. PCR assays revealed diversity at these loci with total allele numbers ranging from 2 to 20, and Nei's diversity index values ranging from 0.36 to 0.93. Cluster analysis identified two genetically distinct groups consistent with the current biovar classification system of F. tularensis. These findings suggest that these VNTR markers are useful for identifying F. tularensis isolates at this taxonomic level. In this study, biovar B isolates were less diverse than those in biovar A, possibly reflecting the history of tularemia in North America. Seven isolates from a recent epizootic in Maricopa County, Ariz., were identical at all VNTR marker loci. Their identity, even at a hypervariable VNTR locus, indicates a common source of infection. This demonstrates the applicability of MLVA for rapid characterization and identification of outbreak isolates. Future construction of reference databases will allow faster outbreak tracking as well as providing a foundation for deciphering global genetic relationships.

BackgroundFrancisella tularensis, the causative agent of tularemia, displays subspecies-specific differences in virulence, geographic distribution, and genetic diversity. F. tularensis subsp. holarctica is widely distributed throughout the Northern Hemisphere. In Europe, F. tularensis subsp. holarctica isolates have largely been assigned to two phylogenetic groups that have specific geographic distributions. Most isolates from Western Europe are assigned to the B.Br.FTNF002-00 group, whereas most isolates from Eastern Europe are assigned to numerous lineages within the B.Br.013 group. The eastern geographic extent of the B.Br.013 group is currently unknown due to a lack of phylogenetic knowledge about populations at the European/Asian juncture and in Asia. In this study, we address this knowledge gap by describing the phylogenetic structure of F. tularensis subsp. holarctica isolates from the country of Georgia, and by placing these isolates into a global phylogeographic context.ResultsWe identified a new genetic lineage of F. tularensis subsp. holarctica from Georgia that belongs to the B.Br.013 group. This new lineage is genetically and geographically distinct from lineages previously described from the B.Br.013 group from Central-Eastern Europe. Importantly, this new lineage is basal within the B.Br.013 group, indicating the Georgian lineage diverged before the diversification of the other known B.Br.013 lineages. Although two isolates from the Georgian lineage were collected nearby in the Ukrainian region of Crimea, all other global isolates assigned to this lineage were collected in Georgia. This restricted geographic distribution, as well as the high levels of genetic diversity within the lineage, is consistent with a relatively older origin and localized differentiation.ConclusionsWe identified a new lineage of F. tularensis subsp. holarctica from Georgia that appears to have an older origin than any other diversified lineages previously described from the B.Br.013 group. This finding suggests that additional phylogenetic studies of F. tularensis subsp. holarctica populations in Eastern Europe and Asia have the potential to yield important new insights into the evolutionary history and phylogeography of this broadly dispersed F. tularensis subspecies.

Human Lyme borreliosis (LB) is the most prevalent arthropod-borne infection in temperate climate zones around the world and is caused by Borrelia spirochetes. We have identified 10 variable-number tandem repeat (VNTR) loci present within the genome of Borrelia burgdorferi and subsequently developed a multiple-locus VNTR analysis (MLVA) typing system for this disease agent. We report here the successful application of MLVA for strain discrimination among a group of 41 globally diverse Borrelia isolates including B. burgdorferi, B. afzelii, and B. garinii. PCR assays displayed diversity at these loci, with total allele numbers ranging from two to nine and Nei's diversity (

We performed whole genome sequencing of a cidofovir {[(S)-1-(3-hydroxy-2-phosphonylmethoxy-propyl) cytosine] [HPMPC]}-resistant (CDV-R) strain of Monkeypoxvirus (MPV). Whole-genome comparison with the wild-type (WT) strain revealed 55 single-nucleotide polymorphisms (SNPs) and one tandem-repeat contraction. Over one-third of all identified SNPs were located within genes comprising the poxvirus replication complex, including the DNA polymerase, RNA polymerase, mRNA capping methyltransferase, DNA processivity factor, and poly-A polymerase. Four polymorphic sites were found within the DNA polymerase gene. DNA polymerase mutations observed at positions 314 and 684 in MPV were consistent with CDV-R loci previously identified in Vaccinia virus (VACV). These data suggest the mechanism of CDV resistance may be highly conserved across Orthopoxvirus (OPV) species. SNPs were also identified within virulence genes such as the A-type inclusion protein, serine protease inhibitor-like protein SPI-3, Schlafen ATPase and thymidylate kinase, among others. Aberrant chain extension induced by CDV may lead to diverse alterations in gene expression and viral replication that may result in both adaptive and attenuating mutations. Defining the potential contribution of substitutions in the replication complex and RNA processing machinery reported here may yield further insight into CDV resistance and may augment current therapeutic development strategies.

BackgroundRecently the genome sequences of two brucellaphages, isolated in Georgia (Tb) and Mexico (Pr) were reported revealing pronounced sequence homogeneity and the presence of two major indels discriminating the two phages. Subsequent genome sequencing of six diagnostic brucellaphages: Tbilisi (Tb), Firenze (Fz), Weybridge (Wb), S708, Berkeley (Bk) and R/C phages identified three major genetic groups. However, the propensity for fine-scale genetic variability of diverse brucellaphages grown on multiple hosts within a single Brucella species remains unknown.MethodsWe sequenced the complete genomes of ten brucellaphages following initial propagation on B. abortus strain 141 and after subsequent propagation on B. abortus strain S19. All brucellaphages were isolated and propagated at the Eliava Institute including the original Tb phage. Genomic libraries were quantified using the Qbit and sheared on the Covaris M220. QC for fragmentation was performed on the BioAnalyzer 2100. DNA libraries were prepared using an Illumina Paired-End protocol and sequenced on the Illumina MiSeq. Sequence analysis was performed using Geneious and MEGA software.ResultsComparative whole genome sequence analysis revealed genetic homogeneity consistent with previously published data as well as multiple nucleotide variations. Genomic changes as a result of passages were observed in similar genes and predominantly occurred at identical sites in separate phages. Multiple instances of within-sample genetic heterogeneity were observed often at shared genomics positions across phages. Positive selection was detected in the tail collar protein gene. We also identified a Staphylothermus marinus F1-like CRISPR spacer and sequences orthologous to both prophage antirepressors of Brucella spp. and intergenic sequences encoded by Ochrobactrum anthropi.ConclusionWe surveyed whole genome level diversity in phage lytic for B. abortus as they are propagated on alternate vaccine strains within the species. Our data extend previous results indicating select variable hotspots and broad genomic homogeneity as well as multiple common polymorphisms and within-sample variation. These data also provide additional genomes for future reference in comparative studies involving the molecular evolution and host specificity of brucellaphages.