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Charles Chiu and colleagues analyze the gut viromes of recipients of hematopoietic stem cell transplantation and identify characteristics associated with the severity of graft-versus-host disease in the gut. Much attention has been focused on the role of the bacterial microbiome in human health, but the virome is understudied. Although previously investigated in individuals with inflammatory bowel diseases or solid-organ transplants 1 , 2 , virome dynamics in allogeneic hematopoietic stem cell transplantation (HSCT) and enteric graft-versus-host disease (GVHD) remain unexplored. Here we characterize the longitudinal gut virome in 44 recipients of HSCT using metagenomics. A viral 'bloom' was identified, and significant increases were demonstrated in the overall proportion of vertebrate viral sequences following transplantation ( P = 0.02). Increases in both the rates of detection ( P < 0.0001) and number of sequences ( P = 0.047) of persistent DNA viruses (anelloviruses, herpesviruses, papillomaviruses and polyomaviruses) over time were observed in individuals with enteric GVHD relative to those without, a finding accompanied by a reduced phage richness ( P = 0.01). Picobirnaviruses were detected in 18 individuals (40.9%), more frequently before or within a week after transplant than at later time points ( P = 0.008). In a time-dependent Cox proportional-hazards model, picobirnaviruses were predictive of the occurrence of severe enteric GVHD (hazard ratio, 2.66; 95% confidence interval (CI) = 1.46–4.86; P = 0.001), and correlated with higher fecal levels of two GVHD severity markers, calprotectin and α1-antitrypsin. These results reveal a progressive expansion of vertebrate viral infections over time following HSCT, and they suggest an unexpected association of picobirnaviruses with early post-transplant GVHD.

Background Chronic obstructive pulmonary disease (COPD) is a heterogeneous disease characterized by frequent exacerbation phenotypes independent of disease stage. Increasing evidence shows that the microbiota plays a role in disease progression and severity, but long-term and international multicenter assessment of the variations in viral and bacterial communities as drivers of exacerbations are lacking. Methods Two-hundred severe COPD patients from Europe and North America were followed longitudinally for 3 years. We performed nucleic acid detection for 20 respiratory viruses and 16S ribosomal RNA gene sequencing to evaluate the bacterial microbiota in 1179 sputum samples collected at stable, acute exacerbation and follow-up visits. Results Similar viral and bacterial taxa were found in patients from the USA compared to Bulgaria and Czech Republic but their microbiome diversity was significantly different ( P  < 0.001) and did not impact exacerbation rates. Virus infection was strongly associated with exacerbation events ( P  < 5E-20). Human rhinovirus (13.1%), coronavirus (5.1%) and influenza virus (3.6%) constitute the top viral pathogens in triggering exacerbation. Moraxella and Haemophilus were 5-fold and 1.6-fold more likely to be the dominating microbiota during an exacerbation event. Presence of Proteobacteria such as Pseudomonas or Staphylococcus amongst others, were associated with exacerbation events (OR > 0.17; P  < 0.02) but more strongly associated with exacerbation frequency (OR > 0.39; P  < 4E-10), as confirmed by longitudinal variations and biotyping of the bacterial microbiota, and suggesting a role of the microbiota in sensitizing the lung. Conclusions This study highlights bacterial taxa in lung sensitization and viral triggers in COPD exacerbations. It provides a global overview of the diverse targets for drug development and explores new microbiome analysis methods to guide future patient management applications.

We report unbiased metagenomic detection of chikungunya virus (CHIKV), Ebola virus (EBOV), and hepatitis C virus (HCV) from four human blood samples by MinION nanopore sequencing coupled to a newly developed, web-based pipeline for real-time bioinformatics analysis on a computational server or laptop (MetaPORE). At titers ranging from 10 7 –10 8 copies per milliliter, reads to EBOV from two patients with acute hemorrhagic fever and CHIKV from an asymptomatic blood donor were detected within 4 to 10 min of data acquisition, while lower titer HCV virus (1 × 10 5 copies per milliliter) was detected within 40 min. Analysis of mapped nanopore reads alone, despite an average individual error rate of 24 % (range 8–49 %), permitted identification of the correct viral strain in all four isolates, and 90 % of the genome of CHIKV was recovered with 97–99 % accuracy. Using nanopore sequencing, metagenomic detection of viral pathogens directly from clinical samples was performed within an unprecedented <6 hr sample-to-answer turnaround time, and in a timeframe amenable to actionable clinical and public health diagnostics.

An increasing number of emerging tick-borne diseases has been reported in the United States since the 1970s. Using metagenomic next generation sequencing, we detected nucleic acid sequences from 2 novel viruses in the family Bunyaviridae and an emerging human rickettsial pathogen, Rickettsia philipii , in a population of the Pacific Coast tick, Dermacentor occidentalis in Mendocino County sampled annually from 2011 to 2014. A total of 250 adults of this human-biting, generalist tick were collected from contiguous chaparral and grassland habitats, and RNA from each individually extracted tick was deep sequenced to an average depth of 7.3 million reads. We detected a Francisella endosymbiont in 174 ticks (70%), and Rickettsia spp . in 19 ticks (8%); Rickettsia-infected ticks contained R. rhipicephali (16 of 250, 6.4%) or R. philipii (3 of 250,1.2%), the agent of eschar-associated febrile illness in humans. The genomes of 2 novel bunyaviruses (>99% complete) in the genera Nairovirus and Phlebovirus were also identified and found to be present in 20–91% of ticks, depending on the year of collection. The high prevalence of these bunyaviruses in sampled Dermacentor ticks suggests that they may be viral endosymbionts, although further studies are needed to determine whether they are infectious for vertebrate hosts, especially humans, and their potential role in tick ecology.

A monkey model of Zika virus (ZIKV) infection is urgently needed to better understand transmission and pathogenesis, given its proven association with fetal brain defects in pregnant women and acute neurological illness. Here we experimentally infected 4 male marmosets with ZIKV (prototype 1947 African strain) and monitored them clinically with sampling of various body fluids and tissues for nearly 3 months. We show that the course of acute infection with ZIKV in these New World monkeys resembles the human illness in many respects, including (1) lack of apparent clinical symptoms in most cases, (2) persistence of the virus in body fluids such as semen and saliva for longer periods of time than in serum, and (3) generation of neutralizing antibodies as well as an antiviral immunological host response. Importantly, ZIKV-infected saliva samples (in addition to serum) were found to be infectious, suggesting potential capacity for viral transmission by the oral route. Re-challenge of a previously infected marmoset with a contemporary outbreak strain SPH2015 from Brazil resulted in continued protection against infection, no viral shedding, and boosting of the immune response. Given the key similarities to human infection, a marmoset model of ZIKV infection may be useful for testing of new drugs and vaccines.

Zika virus (ZIKV) is an emerging, mosquito-borne pathogen associated with a widespread 2015–2016 epidemic in the Western Hemisphere and a proven cause of microcephaly and other fetal brain defects in infants born to infected mothers. ZIKV infections have been also linked to other neurological illnesses in infected adults and children, including Guillain-Barré syndrome (GBS), acute flaccid paralysis (AFP) and meningoencephalitis, but the viral pathophysiology behind those conditions remains poorly understood. Here we investigated ZIKV infectivity in neuroblastoma SH-SY5Y cells, both undifferentiated and following differentiation with retinoic acid. We found that multiple ZIKV strains, representing both the prototype African and contemporary Asian epidemic lineages, were able to replicate in SH-SY5Y cells. Differentiation with resultant expression of mature neuron markers increased infectivity in these cells, and the extent of infectivity correlated with degree of differentiation. New viral particles in infected cells were visualized by electron microscopy and found to be primarily situated inside vesicles; overt damage to the Golgi apparatus was also observed. Enhanced ZIKV infectivity in a neural cell line following differentiation may contribute to viral neuropathogenesis in the developing or mature central nervous system.

Lyme disease is a tick-borne illness caused by the bacterium Borrelia burgdorferi , and approximately 10 to 20% of patients report persistent symptoms lasting months to years despite appropriate treatment with antibiotics. To gain insights into the molecular basis of acute Lyme disease and the ensuing development of post-treatment symptoms, we conducted a longitudinal transcriptome study of 29 Lyme disease patients (and 13 matched controls) enrolled at the time of diagnosis and followed for up to 6 months. The differential gene expression signature of Lyme disease following the acute phase of infection persisted for at least 3 weeks and had fewer than 44% differentially expressed genes (DEGs) in common with other infectious or noninfectious syndromes. Early Lyme disease prior to antibiotic therapy was characterized by marked upregulation of Toll-like receptor signaling but lack of activation of the inflammatory T-cell apoptotic and B-cell developmental pathways seen in other acute infectious syndromes. Six months after completion of therapy, Lyme disease patients were found to have 31 to 60% of their pathways in common with three different immune-mediated chronic diseases. No differential gene expression signature was observed between Lyme disease patients with resolved illness to those with persistent symptoms at 6 months post-treatment. The identification of a sustained differential gene expression signature in Lyme disease suggests that a panel of selected human host-based biomarkers may address the need for sensitive clinical diagnostics during the “window period” of infection prior to the appearance of a detectable antibody response and may also inform the development of new therapeutic targets. IMPORTANCE Lyme disease is the most common tick-borne infection in the United States, and some patients report lingering symptoms lasting months to years despite antibiotic treatment. To better understand the role of the human host response in acute Lyme disease and the development of post-treatment symptoms, we conducted the first longitudinal gene expression (transcriptome) study of patients enrolled at the time of diagnosis and followed up for up to 6 months after treatment. Importantly, we found that the gene expression signature of early Lyme disease is distinct from that of other acute infectious diseases and persists for at least 3 weeks following infection. This study also uncovered multiple previously undescribed pathways and genes that may be useful in the future as human host biomarkers for diagnosis and that constitute potential targets for the development of new therapies. Lyme disease is the most common tick-borne infection in the United States, and some patients report lingering symptoms lasting months to years despite antibiotic treatment. To better understand the role of the human host response in acute Lyme disease and the development of post-treatment symptoms, we conducted the first longitudinal gene expression (transcriptome) study of patients enrolled at the time of diagnosis and followed up for up to 6 months after treatment. Importantly, we found that the gene expression signature of early Lyme disease is distinct from that of other acute infectious diseases and persists for at least 3 weeks following infection. This study also uncovered multiple previously undescribed pathways and genes that may be useful in the future as human host biomarkers for diagnosis and that constitute potential targets for the development of new therapies.

Background Primary amoebic meningoencephalitis (PAM) is a rare, often lethal, cause of encephalitis, for which early diagnosis and prompt initiation of combination antimicrobials may improve clinical outcomes. Methods In this study, we sequenced a full draft assembly of the Balamuthia mandrillaris genome (44.2 Mb in size) from a rare survivor of PAM, and recovered the mitochondrial genome from six additional Balamuthia strains. We also used unbiased metagenomic next-generation sequencing (NGS) and SURPI bioinformatics analysis to diagnose an ultimately fatal case of Balamuthia mandrillaris encephalitis in a 15-year-old girl. Results and Discussion Comparative analysis of the mitochondrial genome and high-copy number genes from six additional Balamuthia mandrillaris strains demonstrated remarkable sequence variation, and the closest Balamuthia homologs corresponded to other amoebae, hydroids, algae, slime molds, and peat moss. Real-time NGS testing of hospital day 6 CSF and brain biopsy samples detected Balamuthia on the basis of high-quality hits to 16S and 18S ribosomal RNA sequences present in the National Center for Biotechnology Information (NCBI) nt reference database. The presumptive diagnosis of PAM by visualization of amoebae on brain biopsy histopathology and NGS analysis was subsequently confirmed at the US Centers for Disease Control and Prevention (CDC) using a Balamuthia -specific PCR assay. Retrospective analysis of a day 1 CSF sample revealed that more timely identification of Balamuthia by metagenomic NGS, potentially resulting in a better clinical outcome, would have required availability of the complete genome sequence. Conclusions These results underscore the diverse evolutionary origins of Balamuthia mandrillaris , provide new targets for diagnostic assay development, and will facilitate further investigations of the biology and pathogenesis of this eukaryotic pathogen. The failure to identify PAM from a day 1 sample without a fully sequenced Balamuthia genome in the database highlights the critical importance of whole-genome reference sequences for microbial detection by metagenomic NGS.

Chronic obstructive pulmonary disease (COPD) is a chronic lung disease characterized by airway obstruction and inflammation. Non-typeable Haemophilus influenzae (NTHi) lung infections are common in COPD, promoting frequent exacerbations and accelerated lung function decline. The relationship with immune responses and NTHi are poorly understood. Herein, we comprehensively characterized the respiratory microbiome and mycobiome of patients while investigating microbial dynamics and host immune changes attributable to NTHi killing activity. Mild-to-moderate COPD patients encompassing frequent and infrequent exacerbators and healthy volunteers (HV) were enrolled. Microbial composition, proteomics and NTHi killing activity was analyzed using bronchoalveolar lavage fluid (BALF). In addition, antigen–antibody titers in sera to COPD pathogens were determined using a multiplex assay. Differential abundance analysis revealed an enrichment of Actinobacteria and Bacteroidetes in the BALF of COPD and HV subjects respectively. Significant differences in the IgA titer response were observed against NTHi antigens in COPD vs. HV. Notably, there was also significantly greater killing activity against NTHi in BALF from COPD vs. HV subjects (OR = 5.64; 95% CI = 1.75–20.20; p  = 0.001). Stratification of COPD patients by NTHi killing activity identified unique microbial and protein signatures wherein Firmicutes , Actinobacteria and haptoglobin were enriched in patients with killing activity. We report that differences in host immune responses and NTHi-killing activity are associated with microbiome changes in mild-to-moderate COPD. This is suggestive of a potential link between the respiratory microbiome and immune activity against NTHi in the context of COPD pathogenesis even at this disease stage.

Myalgic encephalomyelitis / chronic fatigue syndrome (ME/CFS) is a syndrome of unknown etiology characterized by profound fatigue exacerbated by physical activity, also known as post-exertional malaise (PEM). Previously, we did not detect evidence of immune dysregulation or virus reactivation outside of PEM periods. Here we sought to determine whether cardiopulmonary exercise stress testing of ME/CFS patients could trigger such changes. ME/CFS patients (n = 14) and matched sedentary controls (n = 11) were subjected to cardiopulmonary exercise on 2 consecutive days and followed up to 7 days post-exercise, and longitudinal whole blood samples analyzed by RNA-seq. Although ME/CFS patients showed significant worsening of symptoms following exercise versus controls, with 8 of 14 ME/CFS patients showing reduced oxygen consumption ([Formula: see text]) on day 2, transcriptome analysis yielded only 6 differentially expressed gene (DEG) candidates when comparing ME/CFS patients to controls across all time points. None of the DEGs were related to immune signaling, and no DEGs were found in ME/CFS patients before and after exercise. Virome composition (P = 0.746 by chi-square test) and number of viral reads (P = 0.098 by paired t-test) were not significantly associated with PEM. These observations do not support transcriptionally-mediated immune cell dysregulation or viral reactivation in ME/CFS patients during symptomatic PEM episodes.