Biology subjects

Javornik Cregeen, S. J.

Publications and source records attributed to Javornik Cregeen, S. J..

8 recordsLinked to original sources

bioRxiv2026

EsViritu is a sensitive and specific profiler for viral pathogen detection and genome recovery in metagenomes

Metagenomic sequencing allows for detection of any number of pathogenic viruses from a single sample without a priori knowledge of its contents. This has created a substantial opportunity to characterize pathogens in humans and their environment, but the field is held back by a lack of bioinformatics tools for taxonomic profiling and reconstructing virus genomes. The ideal profiler would simultaneously do two things: report viruses at single-read sensitivity so that ultra-low-abundance pathogens are not missed and hold near-perfect specificity so that a pathogen is never falsely reported. Here we present EsViritu, a taxonomic profiler that pairs an iterative greedy clustering algorithm and lowest-common-ancestor taxonomic refinement with a curated Virus Pathogen Database to reconstruct virus genomes and generate virome profiles directly from reads. We benchmarked EsViritu against four leading metagenomic profilers (Kraken2, Centrifuger, Metabuli, and Sylph), running each program across a full parameter sweep and against the same Virus Pathogen Database for an apples-to-apples comparison. Across synthetic positive controls, pathogen-free negative controls, and mutated mock communities, EsViritu was the most accurate tool at reconstructing community composition: it led or tied every tool on relative abundance accuracy at all taxonomic ranks and led or tied on detection across ranks. Importantly, other tools frequently report the presence of multiple related taxa when only reads for a single genome are present, but EsViritu faithfully records the correct, single taxon and genome. EsViritu achieves read-level sensitivity for sequences as low as 85% average nucleotide identity to the nearest reference and reports essentially no false positives in pathogen-free host and synthetic samples. EsViritu also returns reconstructed consensus genomes whose nucleotide identity to the nearest reference closely tracks ground truth, yielding both a profile and genomes from a single run.

bioinformatics↗

bioRxiv2024

Complete Genomic Characterization of Global Pathogens, Respiratory Syncytial Virus (RSV), and Human Norovirus (HuNoV) Using Probe-based Capture Enrichment.

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in children worldwide, while human noroviruses (HuNoV) are a leading cause of epidemic and sporadic acute gastroenteritis. Generating full-length genome sequences for these viruses is crucial for understanding viral diversity and tracking emerging variants. However, obtaining high-quality sequencing data is often challenging due to viral strain variability, quality, and low titers. Here, we present a set of comprehensive oligonucleotide probe sets designed from 1,570 RSV and 1,376 HuNoV isolate sequences in GenBank. Using these probe sets and a capture enrichment sequencing workflow, 85 RSV positive nasal swab samples and 55 (49 stool and six human intestinal enteroids) HuNoV positive samples encompassing major subtypes and genotypes were characterized. The Ct values of these samples ranged from 17.0-29.9 for RSV, and from 20.2-34.8 for HuNoV, with some HuNoV having below the detection limit. The mean percentage of post-processing reads mapped to viral genomes was 85.1% for RSV and 40.8% for HuNoV post-capture, compared to 0.08% and 1.15% in pre-capture libraries, respectively. Full-length genomes were>99% complete in all RSV positive samples and >96% complete in 47/55 HuNoV positive samples--a significant improvement over genome recovery from pre-capture libraries. RSV transcriptome (subgenomic mRNAs) sequences were also characterized from this data. Probe-based capture enrichment offers a comprehensive approach for RSV and HuNoV genome sequencing and monitoring emerging variants. IMPORTANCERespiratory syncytial virus (RSV) and human noroviruses (HuNoV) are NIAID category C and category B priority pathogens, respectively, that inflict significant health consequences on children, adults, immunocompromised patients, and the elderly. Due to the high strain diversity of RSV and HuNoV genomes, obtaining complete genomes to monitor viral evolution and pathogenesis is challenging. In this paper, we present the design, optimization, and benchmarking of a comprehensive oligonucleotide target capture method for these pathogens. All 85 RSV samples and 49/55 HuNoV samples were patient-derived with six human intestinal enteroids. The methodology described here results has a higher success rate in obtaining full-length RSV and HuNoV genomes, enhancing the efficiency of studying these viruses and mutations directly from patient-derived samples.

molecular biology↗

bioRxiv2023

Tryptophan Metabolites And Their Predicted Microbial Sources In Fecal Samples From Healthy Individuals

Gut microbiota produce tryptophan metabolites (TMs) important to homeostasis. However, measuring TM levels in stool and determining their microbial sources can be difficult. Here, we measured TMs from the indole pathway in fecal samples from 21 healthy adults with the goal to: 1) determine fecal TM concentrations in healthy individuals; 2) link TM levels to bacterial abundance using 16S and whole genome shotgun (WGS) sequencing data; and 3) predict likely bacterial sources of TM production. Within our samples, we identified 151 genera (16S) and 592 bacterial species (WGS). Eight TMs were found in [≥]17 fecal samples, including four in all persons. To our knowledge, we are the first to report fecal levels for indole-3-lactate, indole-3-propionate, and 3-indoleacrylate levels in healthy persons. Overall, indole, indole-3-acetate (IAA), and skatole accounted for 86% of the eight TMs measured. Significant correlations were found between seven TMs and 29 bacterial species. Predicted multiple TM sources support the notion of a complex network of TM production and regulation. Further, the data suggest key roles for Collinsella aerofaciens and IAA, a metabolite reported to maintain intestinal homeostasis through enhanced barrier integrity and anti-inflammatory/antioxidant activities. These findings extend our understanding of TMs and their relationship to the microbial species that act as effectors and/or regulators in the healthy intestine and may lead to novel strategies designed to manipulate tryptophan metabolism to prevent disease and/or restore health to the dysbiotic gut. IMPORTANCETryptophan metabolites (TMs) of bacterial origin are increasingly recognized as important signaling molecules among gut microbiota and with the host. However, few reports exist for fecal TM levels in healthy humans, and reported levels vary widely. Further, the specific bacterial species producing TMs and the combinations of fecal TMs in healthy individuals are not well known. Our research combines 16S and whole genome shotgun sequencing of gut bacteria with a sensitive method (LC/MS) for measuring TMs and a reported method to predict which species are likely TM contributors. To our knowledge, this combination of analyses has not been reported elsewhere and will add significantly to the existing literature. Understanding TM levels and their sources in the healthy intestine are fundamental to elucidating how TMs contribute to maintaining homeostasis. Such knowledge of gut microbiota and their metabolic products will inform novel strategies to maintain intestinal health and prevent or treat dysbioses.

microbiology↗

bioRxiv2023

Functional Genomics of Gastrointestinal Escherichia coli Isolated from Patients with Cancer and Diarrhea

We describe the epidemiology and clinical characteristics of 29 patients with cancer and diarrhea in whom Enteroaggregative Escherichia coli (EAEC) was initially identified by GI BioFire panel multiplex. E. coli strains were successfully isolated from fecal cultures in 14 of 29 patients. Six of the 14 strains were identified as EAEC and 8 belonged to other diverse E. coli groups of unknown pathogenesis. We investigated these strains by their adherence to human intestinal organoids, cytotoxic responses, antibiotic resistance profile, full sequencing of their genomes, and annotation of their functional virulome. Interestingly, we discovered novel and enhanced adherence and aggregative patterns for several diarrheagenic pathotypes that were not previously seen when co-cultured with immortalized cell lines. EAEC isolates displayed exceptional adherence and aggregation to human colonoids compared not only to diverse GI E. coli, but also compared to prototype strains of other diarrheagenic E. coli. Some of the diverse E. coli strains that could not be classified as a conventional pathotype also showed an enhanced aggregative and cytotoxic response. Notably, we found a high carriage rate of antibiotic resistance genes in both EAEC strains and diverse GI E. coli isolates and observed a positive correlation between adherence to colonoids and the number of metal acquisition genes carried in both EAEC and the diverse E. coli strains. This work indicates that E. coli from cancer patients constitute strains of remarkable pathotypic and genomic divergence, including strains of unknown disease etiology with unique virulomes. Future studies will allow for the opportunity to re-define E. coli pathotypes with greater diagnostic accuracy and into more clinically relevant groupings.

microbiology↗

bioRxiv2023

INTRA- AND INTER-HOST EVOLUTION OF HUMAN NOROVIRUS IN HEALTHY ADULTS

BackgroundHuman noroviruses are a leading cause of acute and sporadic gastroenteritis worldwide. The evolution of human noroviruses in immunocompromised persons has been evaluated in many studies. Much less is known about the evolutionary dynamics of human norovirus in healthy adults. MethodsWe used sequential samples collected from a controlled human infection study with GI.1/Norwalk/US/68 virus to evaluate intra- and inter-host evolution of a human norovirus in healthy adults. Up to 12 samples from day 1 to day 56 post-challenge were sequenced using a norovirus-specific capture probe method. ResultsComplete genomes were assembled, even in samples that were below the limit of detection of standard RT-qPCR assays, up to 28 days post-challenge. Analysis of 123 complete genomes showed changes in the GI.1 genome in all persons, but there were no conserved changes across all persons. Single nucleotide variants resulting in non-synonymous amino acid changes were observed in all proteins, with the capsid VP1 and nonstructural protein NS3 having the largest numbers of changes. ConclusionsThese data highlight the potential of a new capture-based sequencing approach to assemble human norovirus genomes with high sensitivity and demonstrate limited conserved immune pressure-driven evolution of GI.1 virus in healthy adults.

microbiology↗

bioRxiv2023

Examining intra-host genetic variation of RSV by short read high-throughput sequencing

Every viral infection entails an evolving population of viral genomes. High-throughput sequencing technologies can be used to characterize such populations, but to date there are few published examples of such work. In addition, mixed sequencing data are sometimes used to infer properties of infecting genomes without discriminating between genome-derived reads and reads from the much more abundant, in the case of a typical active viral infection, transcripts. Here we apply capture probe-based short read high-throughput sequencing to nasal wash samples taken from a previously described group of adult hematopoietic cell transplant (HCT) recipients naturally infected with respiratory syncytial virus (RSV). We separately analyzed reads from genomes and transcripts for the levels and distribution of genetic variation by calculating per position Shannon entropies. Our analysis reveals a low level of genetic variation within the RSV infections analyzed here, but with interesting differences between genomes and transcripts in 1) average per sample Shannon entropies; 2) the genomic distribution of variation hotspots; and 3) the genomic distribution of hotspots encoding alternative amino acids. In all, our results suggest the importance of separately analyzing reads from genomes and transcripts when interpreting high-throughput sequencing data for insight into intra-host viral genome replication, expression, and evolution.

genomics↗

bioRxiv2021

Multiple RSV strains infecting HEp-2 and A549 cells reveal cell line-dependent differences in resistance to RSV infection.

Respiratory syncytial virus (RSV) is a leading cause of pediatric acute respiratory infection worldwide. There are currently no approved vaccines or antivirals to combat RSV disease. A few transformed cell lines and two historic strains have been extensively used to study RSV. Here we report a thorough molecular and cell biological characterization of HEp-2 and A549 cells infected with four strains of RSV representing both major subgroups as well as historic and more contemporaneous genotypes -- [RSV/A/Tracy (GA1), RSV/A/Ontario (ON), RSV/B/18537 (GB1), RSV/B/Buenos Aires (BA)] -- via measurements of viral replication kinetics and viral gene expression, immunofluorescence-based imaging of gross cellular morphology and cell-associated RSV, and measurements of host response including transcriptional changes and levels of secreted cytokines and growth factors. Our findings strongly suggest 1) the existence of a conserved difference in gene expression between RSV subgroups A and B; 2) the A549 cell line is a more stringent and natural host of replicating RSV than the HEp-2 cell line; and 3) consistent with previous studies, determining the full effects of viral genetic variation in RSV pathogenesis requires model systems as tractable as transformed cell lines but better representative of the human host. IMPORTANCEInfection with respiratory syncytial virus (RSV) early in life is essentially guaranteed and can lead to severe disease. In vitro data from two historic RSV/A strains and two cell lines, HEp-2 and A549, constitute most of our knowledge; but RSV contains ample variation from two evolving subgroups (A and B) showing recent convergent evolution. Here we measure viral action and host response in HEp-2 and A549 cells infected with four RSV strains from both subgroups and representing both historic and more contemporaneous strains. We discover a subgroup-dependent difference in viral gene expression and find A549 cells are more potently antiviral and more sensitive, albeit subtly, to viral variation. Our findings reveal important differences between RSV subgroups and two widely used cell lines and provide baseline data for experiments with model systems better representative of natural RSV infection.

microbiology↗

bioRxiv2020

Evidence of recombination in coronaviruses implicating pangolin origins of nCoV-2019

A novel coronavirus (nCoV-2019) was the cause of an outbreak of respiratory illness detected in Wuhan, Hubei Province, China in December of 2019. Genomic analyses of nCoV-2019 determined a 96% resemblance with a coronavirus isolated from a bat in 2013 (RaTG13); however, the receptor binding motif (RBM) of these two genomes share low sequence similarity. This divergence suggests a possible alternative source for the RBM coding sequence in nCoV-2019. We identified high sequence similarity in the RBM between nCoV-2019 and a coronavirus genome reconstructed from a viral metagenomic dataset from pangolins possibly indicating a more complex origin for nCoV-2019.

microbiology↗