We applied metagenomic next-generation sequencing (mNGS) to detect Zaire Ebola virus (EBOV) and other potential pathogens from whole-blood samples from 70 patients with suspected Ebola hemorrhagic fever during a 2014 outbreak in Boende, Democratic Republic of the Congo (DRC) and correlated these findings with clinical symptoms. Twenty of 31 patients (64.5%) tested in Kinshasa, DRC, were EBOV positive by quantitative reverse transcriptase PCR (qRT-PCR). Despite partial degradation of sample RNA during shipping and handling, mNGS followed by EBOV-specific capture probe enrichment in a U.S. genomics laboratory identified EBOV reads in 22 of 70 samples (31.4%) versus in 21 of 70 (30.0%) EBOV-positive samples by repeat qRT-PCR (overall concordance = 87.1%). Reads from Plasmodium falciparum (malaria) were detected in 21 patients, of which at least 9 (42.9%) were coinfected with EBOV. Other positive viral detections included hepatitis B virus (n = 2), human pegivirus 1 (n = 2), Epstein-Barr virus (n = 9), and Orungo virus (n = 1), a virus in the Reoviridae family. The patient with Orungo virus infection presented with an acute febrile illness and died rapidly from massive hemorrhage and dehydration. Although the patient’s blood sample was negative by EBOV qRT-PCR testing, identification of viral reads by mNGS confirmed the presence of EBOV coinfection. In total, 9 new EBOV genomes (3 complete genomes, and an additional 6 ≥50% complete) were assembled. Relaxed molecular clock phylogenetic analysis demonstrated a molecular evolutionary rate for the Boende strain 4 to 10× slower than that of other Ebola lineages. These results demonstrate the utility of mNGS in broad-based pathogen detection and outbreak surveillance.
Although the first HIV circulating recombinant form (CRF01_AE) is the predominant strain in many Asian countries, it is uncommonly found in the Congo Basin from where it first originated. To fill the gap in the evolutionary history of this important strain, we sequenced near complete genomes from HIV samples with subgenomic CRF01_AE regions collected in Cameroon and the Democratic Republic of the Congo from 2001 to 2006. HIV genomes were generated from N = 13 plasma specimens by next-generation sequencing of metagenomic libraries prepared with spiked primers targeting HIV, followed by Sanger gap-filling. Genome sequences were aligned to reference strains, including Asian and African CRF01_AE sequences, and evaluated by phylogenetic and recombinant analysis to identify four CRF01_AE strains from Cameroon. We also identified two CRF02, one CRF27, and six unique recombinant form genomes (01jA1jG, 01j02jFjU, FjGj01, A1jDj01, FjGj01, and A1jGj01). Phylogenetic analysis, including the four new African CRF01_AE genomes, placed these samples as a bridge between basal Central African Republic CRF01_AE strains and all Asian, European, and American CRF01_AE strains. Molecular dating confirmed previous estimates indicating that the most recent common CRF01_AE ancestor emerged in the early 1970s (1968-1970) and spread beyond Africa around 1980 to Asia. The new sequences and analysis presented in this study expand the molecular history of the CRF01_AE clade, and are illustrated in an interactive Next Strain phylogenetic tree, map, and timeline at (https://nextstrain.org/community/EduanWilkinson/hiv-1_crf01).
In the version of this Article originally published, the sentence beginning "We also used MPSSE to enrich... " should have stated that the samples were from a patient who developed a fatal viral encephalitis following a 'mosquito bite' rather than a 'tick bite'. The sentence has now been corrected to read: "We also used MSSPE to enrich for Jamestown Canyon virus (JCV) in post-mortem brain tissue samples from a patient who developed a fatal viral encephalitis following a mosquito bite".
BackgroundSince 2014 there have been global biennial outbreaks of acute flaccid myelitis (AFM), a rare but severe “polio-like” illness of as yet-unknown etiology primarily affecting children. Enteroviruses (EVs),, especially EV-D68 and EV-A71, have been implicated in association with AFM cases, but proving causality has been difficult as EVs are rarely isolated from cerebrospinal fluid. In addition, early identification of EV-associated AFM is challenging given that the diagnosis is reliant on potentially subjective clinical and radiological criteria with no specific biomarkers described to date.MethodsWe leveraged existing and newly generated data from a clinical CSF metagenomic assay for pathogen identification at University of California, San Francisco (UCSF) to interrogate the host response at the transcriptome level by RNA sequencing (RNA-Seq). These transcriptome RNA-Seq data were used to create statistical classification models to discriminate among viral infections that have been linked to AFM, including EV-D68, EV-A71, West Nile virus, and Powassan virus. The dynamic range of CSF cellularity (0 to >106 cells/mL), resulting in varying trancriptome coverage, as well as technical variation across samples required the development and validation of novel normalization techniques. In total, we analyzed ~50 CSF samples split into independent training and test sets.ResultsWe were able to demonstrate a distinct signature of AFM that was able to predict the virus associated with AFM in blinded test samples with >80% accuracy. The key transcriptional features that best discriminated EV-A71 from EV-D68-associated AFM involved protein targeting, viral transcription, viral gene expression, and translation initiation pathways.ConclusionHere we demonstrate a novel approach to diagnosis of AFM that relies on host transcriptional biomarkers from cerebrospinal fluid. In the future, this method might allow earlier diagnosis of AFM to drive appropriate therapies and vaccines and predict patient outcomes, as well as guide research studies on the pathophysiology of EV-associated AFM.Disclosures All authors: No reported disclosures.
Metagenomic next-generation sequencing (mNGS) has enabled the high-throughput multiplexed identification of sequences from microbes of potential medical relevance. This approach has become indispensable for viral pathogen discovery and broad-based surveillance of emerging or re-emerging pathogens. From 2015 to 2019, plasma was collected from 9586 individuals in Cameroon and the Democratic Republic of the Congo enrolled in a combined hepatitis virus and retrovirus surveillance program. A subset (n = 726) of the patient specimens was analyzed by mNGS to identify viral co-infections. While co-infections from known blood-borne viruses were detected, divergent sequences from nine poorly characterized or previously uncharacterized viruses were also identified in two individuals. These were assigned to the following groups by genomic and phylogenetic analyses: densovirus, nodavirus, jingmenvirus, bastrovirus, dicistrovirus, picornavirus, and cyclovirus. Although of unclear pathogenicity, these viruses were found circulating at high enough concentrations in plasma for genomes to be assembled and were most closely related to those previously associated with bird or bat excrement. Phylogenetic analyses and in silico host predictions suggested that these are invertebrate viruses likely transmitted through feces containing consumed insects or through contaminated shellfish. This study highlights the power of metagenomics and in silico host prediction in characterizing novel viral infections in susceptible individuals, including those who are immunocompromised from hepatitis viruses and retroviruses, or potentially exposed to zoonotic viruses from animal reservoir species.
Background Pathogens carried by insects, such as Bunyaviruses, are frequently transmitted into human populations and cause disease. Knowing which spillover events represent a public health threat remains a challenge. Metagenomic next-generation sequencing (mNGS) can support infectious disease diagnostics by enabling detection of any pathogen from clinical specimens. Methods mNGS was performed on blood samples to identify potential viral co-infections in HIV+ individuals from Kinshasa, Democratic Republic of Congo (DRC) participating in an HIV diversity cohort study. Time-resolved phylogenetics and molecular assay development assisted in viral characterization. Results The nearly complete genome of a novel orthobunyavirus related to Nyangole virus, a virus previously identified in neighboring Uganda, was assembled from an HBV+ patient. A quantitative PCR assay was designed and used to screen >2,500 plasma samples from Cameroon, DRC, and Uganda, failing to identify any additional cases. Recent sequencing of a US CDC Arbovirus Reference collection revealed that this same virus, now named Bangui virus, was first isolated in 1970 from an individual in the Central African Republic. Time-scaled phylogenetic analyses of Bangui with the related Anopheles and Tanga serogroup complexes indicate that this virus emerged nearly 10,000 years ago. Pervasive and episodic models further suggest this virus is under purifying selection and that only distant common ancestors were subject to positive selection events. Conclusions This study represents only the second identification of a Bangui virus infection in over 50 years. The presumed rarity of Bangui virus infections in humans can be explained by its constraint to an avian host and insect vector, precluding efficient transmission into the human population. Our results demonstrate that molecular phylogenetic analyses can provide insights into the threat posed by novel or re-emergent viruses identified by mNGS.
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