Comparative genomics reveals Cyclospora cayetanensis possesses coccidia-like metabolism and invasion components but unique surface antigens

Assessment of the completeness of sequenced Toxoplasma gondii, Eimeria tenella and Cyclospora cayetanensis genomes based on core eukaryotic protein-encoding genes search using BUSCO. (DOCX 14 kb

Whole-genome analysis of Exserohilum rostratum from an outbreak of fungal meningitis and other infections

Exserohilum rostratum was the cause of most cases of fungal meningitis and other infections associated with the injection of contaminated methylprednisolone acetate produced by the New England Compounding Center (NECC). Until this outbreak, very few human cases of Exserohilum infection had been reported, and very little was known about this dematiaceous fungus, which usually infects plants. Here, we report using whole-genome sequencing (WGS) for the detection of single nucleotide polymorphisms (SNPs) and phylogenetic analysis to investigate the molecular origin of the outbreak using 22 isolates of E. rostratum retrieved from 19 case patients with meningitis or epidural/spinal abscesses, 6 isolates from contaminated NECC vials, and 7 isolates unrelated to the outbreak. Our analysis indicates that all 28 isolates associated with the outbreak had nearly identical genomes of 33.8 Mb. A total of 8 SNPs were detected among the outbreak genomes, with no more than 2 SNPs separating any 2 of the 28 genomes. The outbreak genomes were separated from the next most closely related control strain by ∼136,000 SNPs. We also observed significant genomic variability among strains unrelated to the outbreak, which may suggest the possibility of cryptic speciation in E. rostratum

Development of a new barcode-based, multiplex-PCR, next-generation-sequencing assay and data processing and analytical pipeline for multiplicity of infection detection of Plasmodium falciparum.

BACKGROUND Simultaneous infection with multiple malaria parasite strains is common in high transmission areas. Quantifying the number of strains per host, or the multiplicity of infection (MOI), provides additional parasite indices for assessing transmission levels but it is challenging to measure accurately with current tools. This paper presents new laboratory and analytical methods for estimating the MOI of Plasmodium falciparum. METHODS Based on 24 single nucleotide polymorphisms (SNPs) previously identified as stable, unlinked targets across 12 of the 14 chromosomes within P. falciparum genome, three multiplex PCRs of short target regions and subsequent next generation sequencing (NGS) of the amplicons were developed. A bioinformatics pipeline including B4Screening pathway removed spurious amplicons to ensure consistent frequency calls at each SNP location, compiled amplicons by SNP site diversity, and performed algorithmic haplotype and strain reconstruction. The pipeline was validated by 108 samples generated from cultured-laboratory strain mixtures in different proportions and concentrations, with and without pre-amplification, and using whole blood and dried blood spots (DBS). The pipeline was applied to 273 smear-positive samples from surveys conducted in western Kenya, then providing results into StrainRecon Thresholding for Infection Multiplicity (STIM), a novel MOI estimator. RESULTS The 24 barcode SNPs were successfully identified uniformly across the 12 chromosomes of P. falciparum in a sample using the pipeline. Pre-amplification and parasite concentration, while non-linearly associated with SNP read depth, did not influence the SNP frequency calls. Based on consistent SNP frequency calls at targeted locations, the algorithmic strain reconstruction for each laboratory-mixed sample had 98.5% accuracy in dominant strains. STIM detected up to 5 strains in field samples from western Kenya and showed declining MOI over time (q < 0.02), from 4.32 strains per infected person in 1996 to 4.01, 3.56 and 3.35 in 2001, 2007 and 2012, and a reduction in the proportion of samples with 5 strains from 57% in 1996 to 18% in 2012. CONCLUSION The combined approach of new multiplex PCRs and NGS, the unique bioinformatics pipeline and STIM could identify 24 barcode SNPs of P. falciparum correctly and consistently. The methodology could be applied to field samples to reliably measure temporal changes in MOI

The Phylogenetics and Ecology of the Orthopoxviruses Endemic to North America

The data presented herein support the North American orthopoxviruses (NA OPXV) in a sister relationship to all other currently described Orthopoxvirus (OPXV) species. This phylogenetic analysis reaffirms the identification of the NA OPXV as close relatives of “Old World” (Eurasian and African) OPXV and presents high support for deeper nodes within the Chordopoxvirinae family. The natural reservoir host(s) for many of the described OPXV species remains unknown although a clear virus-host association exists between the genus OPXV and several mammalian taxa. The hypothesized host associations and the deep divergence of the OPXV/NA OPXV clades depicted in this study may reflect the divergence patterns of the mammalian faunas of the Old and New World and reflect a more ancient presence of OPXV on what are now the American continents. Genes from the central region of the poxvirus genome are generally more conserved than genes from either end of the linear genome due to functional constraints imposed on viral replication abilities. The relatively slower evolution of these genes may more accurately reflect the deeper history among the poxvirus group, allowing for robust placement of the NA OPXV within Chordopoxvirinae. Sequence data for nine genes were compiled from three NA OPXV strains plus an additional 50 genomes collected from Genbank. The current, gene sequence based phylogenetic analysis reaffirms the identification of the NA OPXV as the nearest relatives of “Old World” OPXV and presents high support for deeper nodes within the Chordopoxvirinae family. Additionally, the substantial genetic distances that separate the currently described NA OPXV species indicate that it is likely that many more undescribed OPXV/NA OPXV species may be circulating among wild animals in North America

The Bartonella vinsonii subsp. arupensis Immunodominant Surface Antigen BrpA Gene, Encoding a 382-Kilodalton Protein Composed of Repetitive Sequences, Is a Member of a Multigene Family Conserved among Bartonella Species

Bartonella proteins that elicit an antibody response during an infection are poorly defined; therefore, to characterize antigens recognized by the host, a Bartonella genomic expression library was screened with serum from an infected mouse. This process led to the discovery of a Bartonella vinsonii subsp. arupensis gene encoding a 382-kDa protein, part of a gene family encoding large proteins, each containing multiple regions of repetitive segments. The genes were termed brpA to -C (bartonella repeat protein) and bore significant similarity to genes encoding the BadA adhesin protein and members of the variably expressed outer membrane protein family of proteins from Bartonella henselae and Bartonella quintana, respectively

Evaluation of Affymetrix Severe Acute Respiratory Syndrome Resequencing GeneChips in Characterization of the Genomes of Two Strains of Coronavirus Infecting Humans

Severe acute respiratory syndrome (SARS) was discovered during a recent global outbreak of atypical pneumonia. A number of immunologic and molecular studies of the clinical samples led to the conclusion that a novel coronavirus (SARS-CoV) was associated with the outbreak. Later, a SARS resequencing GeneChip was developed by Affymetrix to characterize the complete genome of SARS-CoV on a single GeneChip. The present study was carried out to evaluate the performance of SARS resequencing GeneChips. Two human SARS-CoV strains (CDC#200301157 and Urbani) were resequenced by the SARS GeneChips. Five overlapping PCR amplicons were generated for each strain and hybridized with these GeneChips. The successfully hybridized GeneChips generated nucleotide sequences of nearly complete genomes for the two SARS-CoV strains with an average call rate of 94.6%. Multiple alignments of nucleotide sequences obtained from SARS GeneChips and conventional sequencing revealed full concordance. Furthermore, the GeneChip-based analysis revealed no additional polymorphic sites. The results of this study suggest that GeneChip-based genome characterization is fast and reproducible. Thus, SARS resequencing GeneChips may be employed as an alternate tool to obtain genome sequences of SARS-CoV strains pathogenic for humans in order to further understand the transmission dynamics of these viruses

Draft genome sequence of bacillus cereus LA2007, a human-pathogenic isolate harboring anthrax-like plasmids

We present the genome sequence of Bacillus cereus LA2007, a strain isolated in 2007 from a fatal pneumonia case in Louisiana. Sequence-based genome analysis revealed that LA2007 carries a plasmid highly similar to Bacillus anthracis pXO1, including the genes responsible for the production and regulation of anthrax toxin

Chlamydia psittaci comparative genomics reveals intraspecies variations in the putative outer membrane and type III secretion system genes.

Chlamydia psittaci is an obligate intracellular bacterium that can cause significant disease among a broad range of hosts. In humans, this organism may cause psittacosis, a respiratory disease that can spread to involve multiple organs, and in rare untreated cases may be fatal. There are ten known genotypes based on sequencing the major outer-membrane protein gene, ompA, of C. psittaci. Each genotype has overlapping host preferences and virulence characteristics. Recent studies have compared C. psittaci among other members of the Chlamydiaceae family and showed that this species frequently switches hosts and has undergone multiple genomic rearrangements. In this study, we sequenced five genomes of C. psittaci strains representing four genotypes, A, B, D and E. Due to the known association of the type III secretion system (T3SS) and polymorphic outer-membrane proteins (Pmps) with host tropism and virulence potential, we performed a comparative analysis of these elements among these five strains along with a representative genome from each of the remaining six genotypes previously sequenced. We found significant genetic variation in the Pmps and tbl3SS genes that may partially explain differences noted in C. psittaci host infection and disease