A Model of Cytomegalovirus Association with the Development of Heart Graft Atherosclerosis

The development of graft atherosclerosis in heart transplant recipients has been associated with the development of post-transplant cytomegalovirus infection. The mechanism of this involvement, however, remains unclear. I undertook a study to determine the role of cytomegalovirus in the development of graft atherosclerosis. First, a rapid method of detecting cytomegalovirus based on capillary polymerase chain reaction and gel electrophoresis was employed to determine the presence of cytomegalovirus in a wide variety of clinical samples. Detection of cytomegalovirus is a lengthy procedure under normal conditions, and timely detection of cytomegalovirus in transplant recipients may allow intervention early enough to block the development of graft atherosclerosis. Second, post mortem paraffin embedded coronary artery samples of fifteen heart transplant patients were analyzed by the polymerase chain reaction to determine if chronic latent cytomegalovirus infection of the graft vasculature was responsible for the development of graft atherosclerosis. No patient with graft atherosclerosis had cytomegalovirus positive coronary artery specimens at the time of autopsy. Moreover, one patient who did have cytomegalovirus positive coronary artery specimens had no evidence of graft atherosclerosis at autopsy. Finally, a study was undertaken to determine if cytomegalovirus molecular mimicry of the human leukocyte antigen DR (HLA-DR) beta chain could provide the basis of enhanced immune response against the graft. Complement lysis assays of HLA-DR positive cell lines were performed with a polyclonal serum to the cytomegalovirus antigen (immediate-early-2 antigen) with known five amino acid homology with HLA-DR. Peripheral B cells and a HLA-DR positive cell line were lysed by the serum and complement, while peripheral T cells and a HLA-DR negative cell line were not lysed. Based on these data, a model of cytomegalovirus involvement in the development of graft atherosclerosis is proposed. Cytomegalovirus infection leads to enhanced humoral immunity against graft endothelial cells expressing HLA-DR. Vascular damage to the graft leads to a progressive, proliferative healing response that results in graft atherosclerosis and eventual graft failure

Pandemic (H1N1) 2009 Cluster Analysis: A Preliminary Assessment

Pandemic (H1N1) 2009 virus has been causing major concerns around the world because of its epidemic potential, rapid dissemination, rate of mutations, and the number of fatalities. One way to gain an advantage over this virus is to use existing rapid bioinformatics tools to examine easily and inexpensively generated genetic sequencing data. We have used the protein sequences deposited with the National Center for Biotechnology Information (NCBI) for data mining to study the relationship among the Pandemic (H1N1) 2009 proteins. There are 11 proteins in the Pandemic (H1N1) 2009 virus, and analysis of sequences from 65 different locations around the globe has resulted in two major clusters. These clusters illustrate the Pandemic H1N1 2009 virus is already experiencing significant genetic drift and that rapid worldwide travel is affecting the distribution of genetically distinct isolates

Genomic Signatures of Strain Selection and Enhancement in Bacillus atrophaeus var. globigii, a Historical Biowarfare Simulant

(BG) as a simulant for biological warfare (BW) agents, knowledge of its genome composition is limited. Furthermore, the ability to differentiate signatures of deliberate adaptation and selection from natural variation is lacking for most bacterial agents. We characterized a lineage of BGwith a long history of use as a simulant for BW operations, focusing on classical bacteriological markers, metabolic profiling and whole-genome shotgun sequencing (WGS). on the nucleotide level. WGS of variants revealed that several strains were mixed but highly related populations and uncovered a progressive accumulation of mutations among the “military” isolates. Metabolic profiling and microscopic examination of bacterial cultures revealed enhanced growth of “military” isolates on lactate-containing media, and showed that the “military” strains exhibited a hypersporulating phenotype.Our analysis revealed the genomic and phenotypic signatures of strain adaptation and deliberate selection for traits that were desirable in a simulant organism. Together, these results demonstrate the power of whole-genome and modern systems-level approaches to characterize microbial lineages to develop and validate forensic markers for strain discrimination and reveal signatures of deliberate adaptation

Comparative Genomics of 2009 Seasonal Plague (Yersinia pestis) in New Mexico

Plague disease caused by the Gram-negative bacterium Yersinia pestis routinely affects animals and occasionally humans, in the western United States. The strains native to the North American continent are thought to be derived from a single introduction in the late 19th century. The degree to which these isolates have diverged genetically since their introduction is not clear, and new genomic markers to assay the diversity of North American plague are highly desired. To assay genetic diversity of plague isolates within confined geographic areas, draft genome sequences were generated by 454 pyrosequencing from nine environmental and clinical plague isolates. In silico assemblies of Variable Number Tandem Repeat (VNTR) loci were compared to laboratory-generated profiles for seven markers. High-confidence SNPs and small Insertion/Deletions (Indels) were compared to previously sequenced Y. pestis isolates. The resulting panel of mutations allowed clustering of the strains and tracing of the most likely evolutionary trajectory of the plague strains. The sequences also allowed the identification of new putative SNPs that differentiate the 2009 isolates from previously sequenced plague strains and from each other. In addition, new insertion points for the abundant insertion sequences (IS) of Y. pestis are present that allow additional discrimination of strains; several of these new insertions potentially inactivate genes implicated in virulence. These sequences enable whole-genome phylogenetic analysis and allow the unbiased comparison of closely related isolates of a genetically monomorphic pathogen

Pathosphere.org: pathogen detection and characterization through a web-based, open source informatics platform

Background The detection of pathogens in complex sample backgrounds has been revolutionized by wide access to next-generation sequencing (NGS) platforms. However, analytical methods to support NGS platforms are not as uniformly available. Pathosphere (found at Pathosphere.org) is a cloud - based open - sourced community tool that allows for communication, collaboration and sharing of NGS analytical tools and data amongst scientists working in academia, industry and government. The architecture allows for users to upload data and run available bioinformatics pipelines without the need for onsite processing hardware or technical support. Results The pathogen detection capabilities hosted on Pathosphere were tested by analyzing pathogen-containing samples sequenced by NGS with both spiked human samples as well as human and zoonotic host backgrounds. Pathosphere analytical pipelines developed by Edgewood Chemical Biological Center (ECBC) identified spiked pathogens within a common sample analyzed by 454, Ion Torrent, and Illumina sequencing platforms. ECBC pipelines also correctly identified pathogens in human samples containing arenavirus in addition to animal samples containing flavivirus and coronavirus. These analytical methods were limited in the detection of sequences with limited homology to previous annotations within NCBI databases, such as parvovirus. Utilizing the pipeline-hosting adaptability of Pathosphere, the analytical suite was supplemented by analytical pipelines designed by the United States Army Medical Research Insititute of Infectious Diseases and Walter Reed Army Institute of Research (USAMRIID-WRAIR). These pipelines were implemented and detected parvovirus sequence in the sample that the ECBC iterative analysis previously failed to identify. Conclusions By accurately detecting pathogens in a variety of samples, this work demonstrates the utility of Pathosphere and provides a platform for utilizing, modifying and creating pipelines for a variety of NGS technologies developed to detect pathogens in complex sample backgrounds. These results serve as an exhibition for the existing pipelines and web-based interface of Pathosphere as well as the plug-in adaptability that allows for integration of newer NGS analytical software as it becomes available

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Surface Sampling of Spores in Dry-Deposition Aerosols▿

The ability to reliably and reproducibly sample surfaces contaminated with a biological agent is a critical step in measuring the extent of contamination and determining if decontamination steps have been successful. The recovery operations following the 2001 attacks with Bacillus anthracis spores were complicated by the fact that no standard sample collection format or decontamination procedures were established. Recovery efficiencies traditionally have been calculated based upon biological agents which were applied to test surfaces in a liquid format and then allowed to dry prior to sampling tests, which may not be best suited for a real-world event with aerosolized biological agents. In order to ascertain if differences existed between air-dried liquid deposition and biological spores which were allowed to settle on a surface in a dried format, a study was undertaken to determine if differences existed in surface sampling recovery efficiencies for four representative surfaces. Studies were then undertaken to compare sampling efficiencies between liquid spore deposition and aerosolized spores which were allowed to gradually settle under gravity on four different test coupon types. Tests with both types of deposition compared efficiencies of four unique swabbing materials applied to four surfaces with various surface properties. Our studies demonstrate that recovery of liquid-deposited spores differs significantly from recovery of dry aerosol-deposited spores in most instances. Whether the recovery of liquid-deposited spores is overexaggerated or underrepresented with respect to that of aerosol-deposited spores depends upon the surface material being tested

Shotgun Genome Sequence of a Yersinia enterocolitica Isolate from the Philippines

The first shotgun genome sequence of a microbial pathogen from the Philippines is reported. Yersinia enterocolitica subsp. palearctica strain PhRBD_Ye1 is the first Y. enterocolitica strain sequenced from an animal source, swine, which is a natural source of yersiniosis. The closest phylogenetic match is a human clinical isolate from Germany