Genome sequence of enterovirus D68 from St. Louis, Missouri, USA, 2016

Enterovirus D68 (EV-D68) was rarely observed prior to a widespread outbreak in 2014. We observed its reemergence in St. Louis in 2016 and sequenced the EV-D68 genomes from two patient samples. The 2016 viruses in St. Louis differed from those we had sequenced from the 2014 outbreak but were similar to other viruses circulating nationally in 2016

Whole-genome sequencing of Klebsiella pneumoniae isolates to track strain progression in a single patient with recurrent urinary tract infection

Klebsiella pneumoniae is an important uropathogen that increasingly harbors broad-spectrum antibiotic resistance determinants. Evidence suggests that some same-strain recurrences in women with frequent urinary tract infections (UTIs) may emanate from a persistent intravesicular reservoir. Our objective was to analyze K. pneumoniae isolates collected over weeks from multiple body sites of a single patient with recurrent UTI in order to track ordered strain progression across body sites, as has been employed across patients in outbreak settings. Whole-genome sequencing of 26 K. pneumoniae isolates was performed utilizing the Illumina platform. PacBio sequencing was used to create a refined reference genome of the original urinary isolate (TOP52). Sequence variation was evaluated by comparing the 26 isolate sequences to the reference genome sequence. Whole-genome sequencing of the K. pneumoniae isolates from six different body sites of this patient with recurrent UTI demonstrated 100% chromosomal sequence identity of the isolates, with only a small P2 plasmid deletion in a minority of isolates. No single nucleotide variants were detected. The complete absence of single-nucleotide variants from 26 K. pneumoniae isolates from multiple body sites collected over weeks from a patient with recurrent UTI suggests that, unlike in an outbreak situation with strains collected from numerous patients, other methods are necessary to discern strain progression within a single host over a relatively short time frame.</p

Enhanced virome sequencing using targeted sequence capture

Metagenomic shotgun sequencing (MSS) is an important tool for characterizing viral populations. It is culture independent, requires no a priori knowledge of the viruses in the sample, and may provide useful genomic information. However, MSS can lack sensitivity and may yield insufficient data for detailed analysis. We have created a targeted sequence capture panel, ViroCap, designed to enrich nucleic acid from DNA and RNA viruses from 34 families that infect vertebrate hosts. A computational approach condensed ∼1 billion bp of viral reference sequence into <200 million bp of unique, representative sequence suitable for targeted sequence capture. We compared the effectiveness of detecting viruses in standard MSS versus MSS following targeted sequence capture. First, we analyzed two sets of samples, one derived from samples submitted to a diagnostic virology laboratory and one derived from samples collected in a study of fever in children. We detected 14 and 18 viruses in the two sets, comprising 19 genera from 10 families, with dramatic enhancement of genome representation following capture enrichment. The median fold-increases in percentage viral reads post-capture were 674 and 296. Median breadth of coverage increased from 2.1% to 83.2% post-capture in the first set and from 2.0% to 75.6% in the second set. Next, we analyzed samples containing a set of diverse anellovirus sequences and demonstrated that ViroCap could be used to detect viral sequences with up to 58% variation from the references used to select capture probes. ViroCap substantially enhances MSS for a comprehensive set of viruses and has utility for research and clinical applications

Emerging view of the human virome

The human virome is the collection of all viruses that are found in or on humans, including both eukaryotic and prokaryotic viruses. Eukaryotic viruses clearly have important effects on human health, ranging from mild, self-limited acute or chronic infections to those with serious or fatal consequences. Prokaryotic viruses can also influence human health by affecting bacterial community structure and function. Therefore, definition of the virome is an important step toward understanding how microbes affect human health and disease. We review progress in virome analysis, which has been driven by advances in high-throughput, deep sequencing technology. Highlights from these studies include the association of viruses with clinical phenotypes and description of novel viruses that may be important pathogens. Together these studies indicate that analysis of the human virome is critical as we aim to understand how microbial communities influence human health and disease. Descriptions of the human virome will stimulate future work to understand how the virome affects long-term human health, immunity, and response to coinfections. Analysis of the virome ultimately may affect the treatment of patients with a variety of clinical syndromes

Comparison of metagenomic sequencing and the NanoString nCounter analysis system for the characterization of bacterial and viral communities in vaginal samples

DNA sequencing assays have been used to characterize the vaginal microbiome and to identify associations with clinical outcomes. The purpose of this study was to evaluate the utility of the NanoString nCounter platform, a more efficient assay compared to sequencing, for the characterization of vaginal microbial communities. A panel of NanoString nCounter probes was designed to detect common vaginal bacteria and viruses with relevance to reproductive health. A defined synthetic community of microbes and 43 clinical samples were interrogated with NanoString nCounter assays and compared to known compositions or metagenomic shotgun sequencing (MSS) results. The NanoString nCounter platform and MSS were able to distinguish closely related microbes. In clinical samples, the relative abundance of bacterial species was similar between the two assays. The assays sometimes disagreed when targets were present at low abundance. More viruses were detected by MSS than by nCounter. However, the nCounter assays are able to provide results in about 30 h with minimal hands-on time, whereas MSS requires at least 138 to 178 h with extensive hands-on time. The reagent cost for the two assays was similar, but the overall cost of the nCounter was lower due to the minimal hands-on time. MSS can be used to inform the design of a targeted multiplex panel for the assessment of vaginal microbial communities, thereby allowing for more cost-effective and rapid screening of patient samples for research studies. The sensitivity for low abundance microbes could be improved, possibly by adding additional target amplification cycles before nCounter assessment. This approach has potential as an assay with both research and clinical applications

Development and evaluation of an enterovirus D68 real-time reverse transcriptase PCR assay

We have developed and evaluated a real-time reverse transcriptase PCR (RT-PCR) assay for the detection of human enterovirus D68 (EV-D68) in clinical specimens. This assay was developed in response to the unprecedented 2014 nationwide EV-D68 outbreak in the United States associated with severe respiratory illness. As part of our evaluation of the outbreak, we sequenced and published the genome sequence of the EV-D68 virus circulating in St. Louis, MO. This sequence, along with other GenBank sequences from past EV-D68 occurrences, was used to computationally select a region of EV-D68 appropriate for targeting in a strain-specific RT-PCR assay. The RT-PCR assay amplifies a segment of the VP1 gene, with an analytic limit of detection of 4 copies per reaction, and it was more sensitive than commercially available assays that detect enteroviruses and rhinoviruses without distinguishing between the two, including three multiplex respiratory panels approved for clinical use by the FDA. The assay did not detect any other enteroviruses or rhinoviruses tested and did detect divergent strains of EV-D68, including the first EV-D68 strain (Fermon) identified in California in 1962. This assay should be useful for identifying and studying current and future outbreaks of EV-D68 viruses

Whole-Genome Sequencing of Klebsiella pneumoniae Isolates to Track Strain Progression in a Single Patient With Recurrent Urinary Tract Infection

Klebsiella pneumoniae is an important uropathogen that increasingly harbors broad-spectrum antibiotic resistance determinants. Evidence suggests that some same-strain recurrences in women with frequent urinary tract infections (UTIs) may emanate from a persistent intravesicular reservoir. Our objective was to analyze K. pneumoniae isolates collected over weeks from multiple body sites of a single patient with recurrent UTI in order to track ordered strain progression across body sites, as has been employed across patients in outbreak settings. Whole-genome sequencing of 26 K. pneumoniae isolates was performed utilizing the Illumina platform. PacBio sequencing was used to create a refined reference genome of the original urinary isolate (TOP52). Sequence variation was evaluated by comparing the 26 isolate sequences to the reference genome sequence. Whole-genome sequencing of the K. pneumoniae isolates from six different body sites of this patient with recurrent UTI demonstrated 100% chromosomal sequence identity of the isolates, with only a small P2 plasmid deletion in a minority of isolates. No single nucleotide variants were detected. The complete absence of single-nucleotide variants from 26 K. pneumoniae isolates from multiple body sites collected over weeks from a patient with recurrent UTI suggests that, unlike in an outbreak situation with strains collected from numerous patients, other methods are necessary to discern strain progression within a single host over a relatively short time frame

Metagenomic analysis of double-stranded DNA viruses in healthy adults

BackgroundThe Human Microbiome Project (HMP) was undertaken with the goal of defining microbial communities in and on the bodies of healthy individuals using high-throughput, metagenomic sequencing analysis. The viruses present in these microbial communities, the `human virome¿, are an important aspect of the human microbiome that is particularly understudied in the absence of overt disease. We analyzed eukaryotic double-stranded DNA (dsDNA) viruses, together with dsDNA replicative intermediates of single-stranded DNA viruses, in metagenomic sequence data generated by the HMP. 706 samples from 102 subjects were studied, with each subject sampled at up to five major body habitats: nose, skin, mouth, vagina, and stool. Fifty-one individuals had samples taken at two or three time points 30 to 359 days apart from at least one of the body habitats.ResultsWe detected an average of 5.5 viral genera in each individual. At least 1 virus was detected in 92% of the individuals sampled. These viruses included herpesviruses, papillomaviruses, polyomaviruses, adenoviruses, anelloviruses, parvoviruses, and circoviruses. Each individual had a distinct viral profile, demonstrating the high interpersonal diversity of the virome. Some components of the virome were stable over time.ConclusionsThis study is the first to use high-throughput DNA sequencing to describe the diversity of eukaryotic dsDNA viruses in a large cohort of normal individuals who were sampled at multiple body sites. Our results show that the human virome is a complex component of the microbial flora. Some viruses establish long-term infections that may be associated with increased risk or possibly with protection from disease. A better understanding of the composition and dynamics of the virome may hold important keys to human health. BMC Biol 2014 Sep 10; 12(1):71

The plasma virome in longitudinal samples from pregnant patients

INTRODUCTION: Nucleic acid from viruses is common in peripheral blood, even in asymptomatic individuals. How physiologic changes of pregnancy impact host-virus dynamics for acute, chronic, and latent viral infections is not well described. Previously we found higher viral diversity in the vagina during pregnancy associated with preterm birth (PTB) and Black race. We hypothesized that higher diversity and viral copy numbers in the plasma would show similar trends. METHODS: To test this hypothesis, we evaluated longitudinally collected plasma samples from 23 pregnant patients (11 term and 12 preterm) using metagenomic sequencing with ViroCap enrichment to enhance virus detection. Sequence data were analyzed with the ViroMatch pipeline. RESULTS: We detected nucleic acid from at least 1 virus in at least 1 sample from 87% (20/23) of the maternal subjects. The viruses represented 5 families: DISCUSSION: These results emphasize the importance of longitudinal sampling and diverse cohorts in studies of virome dynamics during pregnancy