Influenza is a major global public health threat as a result of its highly pathogenic variants, large zoonotic reservoir, and pandemic potential. Metagenomic viral sequencing offers the potential for a diagnostic test for influenza virus which also provides insights on transmission, evolution, and drug resistance and simultaneously detects other viruses. We therefore set out to apply the Oxford Nanopore Technologies sequencing method to metagenomic sequencing of respiratory samples. We generated influenza virus reads down to a limit of detection of 10 2 to 10 3 genome copies/ml in pooled samples, observing a strong relationship between the viral titer and the proportion of influenza virus reads (P ϭ 4.7 ϫ 10 Ϫ5 ). Applying our methods to clinical throat swabs, we generated influenza virus reads for 27/27 samples with mid-to-high viral titers (cycle threshold [C T ] values, Ͻ30) and 6/13 samples with low viral titers (C T values, 30 to 40). No false-positive reads were generated from 10 influenza virus-negative samples. Thus, Nanopore sequencing operated with 83% sensitivity (95% confidence interval [CI], 67 to 93%) and 100% specificity (95% CI, 69 to 100%) compared to the current diagnostic standard. Coverage of full-length virus was dependent on sample composition, being negatively influenced by increased host and bacterial reads. However, at high influenza virus titers, we were able to reconstruct Ͼ99% complete sequences for all eight gene segments. We also detected a human coronavirus coinfection in one clinical sample. While further optimization is required to improve sensitivity, this approach shows promise for the Nanopore platform to be used in the diagnosis and genetic analysis of influenza virus and other respiratory viruses.
The poor performance of 2014–15 Northern Hemisphere (NH) influenza vaccines was attributed to mismatched H3N2 component with circulating epidemic strains. Using human serum samples collected from 2009–10, 2010–11 and 2014–15 NH influenza vaccine trials, we assessed their cross-reactive hemagglutination inhibition (HAI) antibody responses against recent H3 epidemic isolates. All three populations (children, adults, and older adults) vaccinated with the 2014–15 NH egg- or cell-based vaccine, showed >50% reduction in HAI post-vaccination geometric mean titers against epidemic H3 isolates from those against egg-grown H3 vaccine strain A/Texas/50/2012 (TX/12e). The 2014–15 NH vaccines, regardless of production type, failed to further extend HAI cross-reactivity against H3 epidemic strains from previous seasonal vaccines. Head-to-head comparison between ferret and human antisera derived antigenic maps revealed different antigenic patterns among representative egg- and cell-grown H3 viruses characterized. Molecular modeling indicated that the mutations of epidemic H3 strains were mainly located in antibody-binding sites A and B as compared with TX/12e. To improve vaccine strain selection, human serologic testing on vaccination-induced cross-reactivity need be emphasized along with virus antigenic characterization by ferret model.
Metagenomic sequencing with the Oxford Nanopore MinION sequencer offers potential for point-of-care testing of infectious diseases in clinical settings. To improve cost-effectiveness, multiplexing of several, barcoded samples upon a single flow cell will be required during sequencing. We generated a unique sequencing dataset to assess the extent and source of cross barcode contamination caused by multiplex MinION sequencing. Sequencing libraries for three different viruses, including influenza A, dengue, and chikungunya, were prepared separately and sequenced on individual flow cells. We also pooled the respective libraries and performed multiplex sequencing. We identified 0.056% of total reads in the multiplex sequencing data that were assigned to incorrect barcodes. Chimeric reads were the predominant source of this error. Our findings highlight the need for careful filtering of multiplex sequencing data before downstream analysis, and the trade-off between sensitivity and specificity that applies to the barcode demultiplexing methods.
To determine whether, and to what extent, influenza A subtype H3 viruses were present in feral swine in the United States, we conducted serologic and virologic surveillance during October 2011–September 2012. These animals were periodically exposed to and infected with A(H3N2) viruses, suggesting they may threaten human and animal health.
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