Charles Y. Chiu

Actionable Diagnosis of Neuroleptospirosis by Next-Generation Sequencing

A 14-year-old boy with severe combined immunodeficiency presented three times to a medical facility over a period of 4 months with fever and headache that progressed to hydrocephalus and status epilepticus necessitating a medically induced coma. Diagnostic workup including brain biopsy was unrevealing. Unbiased next-generation sequencing of the cerebrospinal fluid identified 475 of 3,063,784 sequence reads (0.016%) corresponding to leptospira infection. Clinical assays for leptospirosis were negative. Targeted antimicrobial agents were administered, and the patient was discharged home 32 days later with a status close to his premorbid condition. Polymerase-chain-reaction (PCR) and serologic testing at the Centers for Disease Control and Prevention (CDC) subsequently confirmed evidence of Leptospira santarosai infection.

Recovery of divergent avian bornaviruses from cases of proventricular dilatation disease: Identification of a candidate etiologic agent

BackgroundProventricular dilatation disease (PDD) is a fatal disorder threatening domesticated and wild psittacine birds worldwide. It is characterized by lymphoplasmacytic infiltration of the ganglia of the central and peripheral nervous system, leading to central nervous system disorders as well as disordered enteric motility and associated wasting. For almost 40 years, a viral etiology for PDD has been suspected, but to date no candidate etiologic agent has been reproducibly linked to the disease.ResultsAnalysis of 2 PDD case-control series collected independently on different continents using a pan-viral microarray revealed a bornavirus hybridization signature in 62.5% of the PDD cases (5/8) and none of the controls (0/8). Ultra high throughput sequencing was utilized to recover the complete viral genome sequence from one of the virus-positive PDD cases. This revealed a bornavirus-like genome organization for this agent with a high degree of sequence divergence from all prior bornavirus isolates. We propose the name avian bornavirus (ABV) for this agent. Further specific ABV PCR analysis of an additional set of independently collected PDD cases and controls yielded a significant difference in ABV detection rate among PDD cases (71%, n = 7) compared to controls (0%, n = 14) (P = 0.01; Fishers Exact Test). Partial sequence analysis of a total of 16 ABV isolates we have now recovered from these and an additional set of cases reveals at least 5 distinct ABV genetic subgroups.ConclusionThese studies clearly demonstrate the existence of an avian reservoir of remarkably diverse bornaviruses and provide a compelling candidate in the search for an etiologic agent of PDD.

Cryo-EM visualization of an exposed RGD epitope on adenovirus that escapes antibody neutralization

Interaction of the adenovirus penton base protein with αV integrins promotes virus entry into host cells. The location of the integrin binding sequence Arg‐Gly‐Asp (RGD) on human type 2 adenovirus (Ad2) was visualized by cryo‐electron microscopy (cryo‐EM) and image reconstruction using a mAb (DAV‐1) which recognizes a linear epitope, IRGDTFATR. The sites for DAV‐1 binding corresponded to the weak density above each of the five 22 Å protrusions on the adenovirus penton base protein. Modeling of a Fab fragment crystal structure into the adenovirus‐Fab cryo‐EM density indicated a large amplitude of motion for the Fab and the RGD epitope. An unexpected finding was that Fab fragments, but not IgG antibody molecules, inhibited adenovirus infection. Steric hindrance from the adenovirus fiber and a few bound IgG molecules, as well as epitope mobility, most likely prevent binding of IgG antibodies to all five RGD sites on the penton base protein within the intact virus. These studies indicate that the structure of the adenovirus particle facilitates interaction with cell integrins, whilst restricting binding of potentially neutralizing antibodies.

A novel outbreak enterovirus D68 strain associated with acute flaccid myelitis cases in the USA (2012–14): a retrospective cohort study

BACKGROUND Enterovirus D68 was implicated in a widespread outbreak of severe respiratory illness across the USA in 2014 and has also been reported sporadically in patients with acute flaccid myelitis. We aimed to investigate the association between enterovirus D68 infection and acute flaccid myelitis during the 2014 enterovirus D68 respiratory outbreak in the USA. METHODS Patients with acute flaccid myelitis who presented to two hospitals in Colorado and California, USA, between Nov 24, 2013, and Oct 11, 2014, were included in the study. Additional cases identified from Jan 1, 2012, to Oct 4, 2014, via statewide surveillance were provided by the California Department of Public Health. We investigated the cause of these cases by metagenomic next-generation sequencing, viral genome recovery, and enterovirus D68 phylogenetic analysis. We compared patients with acute flaccid myelitis who were positive for enterovirus D68 with those with acute flaccid myelitis but negative for enterovirus D68 using the two-tailed Fishers exact test, two-sample unpaired t test, and Mann-Whitney U test. FINDINGS 48 patients were included: 25 with acute flaccid myelitis, two with enterovirus-associated encephalitis, five with enterovirus-D68-associated upper respiratory illness, and 16 with aseptic meningitis or encephalitis who tested positive for enterovirus. Enterovirus D68 was detected in respiratory secretions from seven (64%) of 11 patients comprising two temporally and geographically linked acute flaccid myelitis clusters at the height of the 2014 outbreak, and from 12 (48%) of 25 patients with acute flaccid myelitis overall. Phylogenetic analysis revealed that all enterovirus D68 sequences associated with acute flaccid myelitis grouped into a clade B1 strain that emerged in 2010. Of six coding polymorphisms in the clade B1 enterovirus D68 polyprotein, five were present in neuropathogenic poliovirus or enterovirus D70, or both. One child with acute flaccid myelitis and a sibling with only upper respiratory illness were both infected by identical enterovirus D68 strains. Enterovirus D68 viraemia was identified in a child experiencing acute neurological progression of his paralytic illness. Deep metagenomic sequencing of cerebrospinal fluid from 14 patients with acute flaccid myelitis did not reveal evidence of an alternative infectious cause to enterovirus D68. INTERPRETATION These findings strengthen the putative association between enterovirus D68 and acute flaccid myelitis and the contention that acute flaccid myelitis is a rare yet severe clinical manifestation of enterovirus D68 infection in susceptible hosts. FUNDING National Institutes of Health, University of California, Abbott Laboratories, and the Centers for Disease Control and Prevention.

Identification of cardioviruses related to Theiler's murine encephalomyelitis virus in human infections

Cardioviruses comprise a genus of picornaviruses that cause severe illnesses in rodents, but little is known about the prevalence, diversity, or spectrum of disease of such agents among humans. A single cardiovirus isolate, Saffold virus, was cultured in 1981 in stool from an infant with fever. Here, we describe the identification of a group of human cardioviruses that have been cloned directly from patient specimens, the first of which was detected using a pan-viral microarray in respiratory secretions from a child with influenza-like illness. Phylogenetic analysis of the nearly complete viral genome (7961 bp) revealed that this virus belongs to the Theilers murine encephalomyelitis virus (TMEV) subgroup of cardioviruses and is most closely related to Saffold virus. Subsequent screening by RT-PCR of 719 additional respiratory specimens [637 (89%) from patients with acute respiratory illness] and 400 cerebrospinal fluid specimens from patients with neurological disease (aseptic meningitis, encephalitis, and multiple sclerosis) revealed no evidence of cardiovirus infection. However, screening of 751 stool specimens from 498 individuals in a gastroenteritis cohort resulted in the detection of 6 additional cardioviruses (1.2%). Although all 8 human cardioviruses (including Saffold virus) clustered together by phylogenetic analysis, significant sequence diversity was observed in the VP1 gene (66.9%–100% pairwise amino acid identities). These findings suggest that there exists a diverse group of novel human Theilers murine encephalomyelitis virus-like cardioviruses that hitherto have gone largely undetected, are found primarily in the gastrointestinal tract, can be shed asymptomatically, and have potential links to enteric and extraintestinal disease.

The complete genome of klassevirus - a novel picornavirus in pediatric stool.

BackgroundDiarrhea kills 2 million children worldwide each year, yet an etiological agent is not found in approximately 30–50% of cases. Picornaviral genera such as enterovirus, kobuvirus, cosavirus, parechovirus, hepatovirus, teschovirus, and cardiovirus have all been found in human and animal diarrhea. Modern technologies, especially deep sequencing, allow rapid, high-throughput screening of clinical samples such as stool for new infectious agents associated with human disease.ResultsA pool of 141 pediatric gastroenteritis samples that were previously found to be negative for known diarrheal viruses was subjected to pyrosequencing. From a total of 937,935 sequence reads, a collection of 849 reads distantly related to Aichi virus were assembled and found to comprise 75% of a novel picornavirus genome. The complete genome was subsequently cloned and found to share 52.3% nucleotide pairwise identity and 38.9% amino acid identity to Aichi virus. The low level of sequence identity suggests a novel picornavirus genus which we have designated klassevirus. Blinded screening of 751 stool specimens from both symptomatic and asymptomatic individuals revealed a second positive case of klassevirus infection, which was subsequently found to be from the index cases 11-month old twin.ConclusionWe report the discovery of human klassevirus 1, a member of a novel picornavirus genus, in stool from two infants from Northern California. Further characterization and epidemiological studies will be required to establish whether klasseviruses are significant causes of human infection.

A metagenomic analysis of pandemic influenza A (2009 H1N1) infection in patients from North America.

Although metagenomics has been previously employed for pathogen discovery, its cost and complexity have prevented its use as a practical front-line diagnostic for unknown infectious diseases. Here we demonstrate the utility of two metagenomics-based strategies, a pan-viral microarray (Virochip) and deep sequencing, for the identification and characterization of 2009 pandemic H1N1 influenza A virus. Using nasopharyngeal swabs collected during the earliest stages of the pandemic in Mexico, Canada, and the United States (n = 17), the Virochip was able to detect a novel virus most closely related to swine influenza viruses without a priori information. Deep sequencing yielded reads corresponding to 2009 H1N1 influenza in each sample (percentage of aligned sequences corresponding to 2009 H1N1 ranging from 0.0011% to 10.9%), with up to 97% coverage of the influenza genome in one sample. Detection of 2009 H1N1 by deep sequencing was possible even at titers near the limits of detection for specific RT-PCR, and the percentage of sequence reads was linearly correlated with virus titer. Deep sequencing also provided insights into the upper respiratory microbiota and host gene expression in response to 2009 H1N1 infection. An unbiased analysis combining sequence data from all 17 outbreak samples revealed that 90% of the 2009 H1N1 genome could be assembled de novo without the use of any reference sequence, including assembly of several near full-length genomic segments. These results indicate that a streamlined metagenomics detection strategy can potentially replace the multiple conventional diagnostic tests required to investigate an outbreak of a novel pathogen, and provide a blueprint for comprehensive diagnosis of unexplained acute illnesses or outbreaks in clinical and public health settings.

The Perils of Pathogen Discovery: Origin of a Novel Parvovirus-Like Hybrid Genome Traced to Nucleic Acid Extraction Spin Columns

ABSTRACT Next-generation sequencing was used for discovery and de novo assembly of a novel, highly divergent DNA virus at the interface between the Parvoviridae and Circoviridae. The virus, provisionally named parvovirus-like hybrid virus (PHV), is nearly identical by sequence to another DNA virus, NIH-CQV, previously detected in Chinese patients with seronegative (non-A-E) hepatitis. Although we initially detected PHV in a wide range of clinical samples, with all strains sharing ∼99% nucleotide and amino acid identity with each other and with NIH-CQV, the exact origin of the virus was eventually traced to contaminated silica-binding spin columns used for nucleic acid extraction. Definitive confirmation of the origin of PHV, and presumably NIH-CQV, was obtained by in-depth analyses of water eluted through contaminated spin columns. Analysis of environmental metagenome libraries detected PHV sequences in coastal marine waters of North America, suggesting that a potential association between PHV and diatoms (algae) that generate the silica matrix used in the spin columns may have resulted in inadvertent viral contamination during manufacture. The confirmation of PHV/NIH-CQV as laboratory reagent contaminants and not bona fide infectious agents of humans underscores the rigorous approach needed to establish the validity of new viral genomes discovered by next-generation sequencing.

Viral Infection in Acute Exacerbation of Idiopathic Pulmonary Fibrosis

RATIONALE Idiopathic pulmonary fibrosis is a progressive, uniformly fatal interstitial lung disease. An acute exacerbation of idiopathic pulmonary fibrosis is an episode of acute respiratory worsening without an identifiable etiology. Occult viral infection has been proposed as a possible cause of acute exacerbation. OBJECTIVES To use unbiased genomics-based discovery methods to define the role of viruses in acute exacerbation of idiopathic pulmonary fibrosis. METHODS Bronchoalveolar lavage and serum from patients with acute exacerbation of idiopathic pulmonary fibrosis, stable disease, and acute lung injury were tested for viral nucleic acid using multiplex polymerase chain reaction, pan-viral microarray, and high-throughput cDNA sequencing. MEASUREMENTS AND MAIN RESULTS Four of forty-three patients with acute exacerbation of idiopathic pulmonary fibrosis had evidence of common respiratory viral infection (parainfluenza [n = 1], rhinovirus [n = 2], coronavirus [n = 1]); no viruses were detected in the bronchoalveolar lavage from stable patients. Pan-viral microarrays revealed additional evidence of viral infection (herpes simplex virus [n = 1], Epstein-Barr virus [n = 2], and torque teno virus [TTV] [n = 12]) in patients with acute exacerbation. TTV infection was significantly more common in patients with acute exacerbation than stable controls (P = 0.0003), but present in a similar percentage of acute lung injury controls. Deep sequencing of a subset of acute exacerbation cases confirmed the presence of TTV but did not identify additional viruses. CONCLUSIONS Viral infection was not detected in most cases of acute exacerbation of idiopathic pulmonary fibrosis. TTV was present in a significant minority of cases, and cases of acute lung injury; the clinical significance of this finding remains to be determined.

Toward an Understanding of Changes in Diversity Associated with Fecal Microbiome Transplantation Based on 16S rRNA Gene Deep Sequencing

ABSTRACT Fecal microbiome transplantation by low-volume enema is an effective, safe, and inexpensive alternative to antibiotic therapy for patients with chronic relapsing Clostridium difficile infection (CDI). We explored the microbial diversity of pre- and posttransplant stool specimens from CDI patients (n = 6) using deep sequencing of the 16S rRNA gene. While interindividual variability in microbiota change occurs with fecal transplantation and vancomycin exposure, in this pilot study we note that clinical cure of CDI is associated with an increase in diversity and richness. Genus- and species-level analysis may reveal a cocktail of microorganisms or products thereof that will ultimately be used as a probiotic to treat CDI. IMPORTANCE Antibiotic-associated diarrhea (AAD) due to Clostridium difficile is a widespread phenomenon in hospitals today. Despite the use of antibiotics, up to 30% of patients are unable to clear the infection and suffer recurrent bouts of diarrheal disease. As a result, clinicians have resorted to fecal microbiome transplantation (FT). Donor stool for this type of therapy is typically obtained from a spouse or close relative and thoroughly tested for various pathogenic microorganisms prior to infusion. Anecdotal reports suggest a very high success rate of FT in patients who fail antibiotic treatment (>90%). We used deep-sequencing technology to explore the human microbial diversity in patients with Clostridium difficile infection (CDI) disease after FT. Genus- and species-level analysis revealed a cocktail of microorganisms in the Bacteroidetes and Firmicutes phyla that may ultimately be used as a probiotic to treat CDI. Antibiotic-associated diarrhea (AAD) due to Clostridium difficile is a widespread phenomenon in hospitals today. Despite the use of antibiotics, up to 30% of patients are unable to clear the infection and suffer recurrent bouts of diarrheal disease. As a result, clinicians have resorted to fecal microbiome transplantation (FT). Donor stool for this type of therapy is typically obtained from a spouse or close relative and thoroughly tested for various pathogenic microorganisms prior to infusion. Anecdotal reports suggest a very high success rate of FT in patients who fail antibiotic treatment (>90%). We used deep-sequencing technology to explore the human microbial diversity in patients with Clostridium difficile infection (CDI) disease after FT. Genus- and species-level analysis revealed a cocktail of microorganisms in the Bacteroidetes and Firmicutes phyla that may ultimately be used as a probiotic to treat CDI.