A novel twelve class fluctuation test reveals higher than expected mutation rates for influenza A viruses

  1. Matthew D Pauly
  2. Megan C Procario
  3. Adam S Lauring  Is a corresponding author
  1. University of Michigan, United States

Abstract

Influenza virus' low replicative fidelity contributes to its capacity for rapid evolution. Clonal sequencing and fluctuation tests have suggested that the influenza virus mutation rate is 2.7x10-6 - 3.0x10-5 substitutions per nucleotide per strand copied (s/n/r). However, sequencing assays are biased toward mutations with minimal fitness impacts and fluctuation tests typically investigate only a subset of all possible single nucleotide mutations. We developed a fluctuation test based on reversion to fluorescence in a set of virally encoded mutant green fluorescent proteins, which allowed us to measure the rates of selectively neutral mutations representative of the twelve different mutation types. We measured an overall mutation rate of 1.8x10-4 s/n/r for PR8 (H1N1) and 2.5x10-4 s/n/r for Hong Kong 2014 (H3N2) and a transitional bias of 2.7-3.6. Our data suggest that each replicated genome will have an average of 2-3 mutations and highlight the importance of mutational load in influenza virus evolution.

Article and author information

Author details

  1. Matthew D Pauly

    Department of Microbiology and Immunology, University of Michigan, Ann Arbor, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Megan C Procario

    Department of Microbiology and Immunology, University of Michigan, Ann Arbor, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Adam S Lauring

    Department of Microbiology and Immunology, University of Michigan, Ann Arbor, United States
    For correspondence
    alauring@med.umich.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2906-8335

Funding

National Institute of Allergy and Infectious Diseases (R01 AI118886)

  • Adam S Lauring

Doris Duke Charitable Foundation (CSDA 2013105)

  • Adam S Lauring

National Institute of General Medical Sciences (T32 GM007544)

  • Matthew D Pauly

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Copyright

© 2017, Pauly et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 5,747
    views
  • 800
    downloads
  • 116
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Matthew D Pauly
  2. Megan C Procario
  3. Adam S Lauring
(2017)
A novel twelve class fluctuation test reveals higher than expected mutation rates for influenza A viruses
eLife 6:e26437.
https://doi.org/10.7554/eLife.26437

Share this article

https://doi.org/10.7554/eLife.26437

Further reading

    1. Genetics and Genomics
    2. Microbiology and Infectious Disease
    Iti Mehta, Jacob B Hogins ... Larry Reitzer
    Research Article

    Polyamines are biologically ubiquitous cations that bind to nucleic acids, ribosomes, and phospholipids and, thereby, modulate numerous processes, including surface motility in Escherichia coli. We characterized the metabolic pathways that contribute to polyamine-dependent control of surface motility in the commonly used strain W3110 and the transcriptome of a mutant lacking a putrescine synthetic pathway that was required for surface motility. Genetic analysis showed that surface motility required type 1 pili, the simultaneous presence of two independent putrescine anabolic pathways, and modulation by putrescine transport and catabolism. An immunological assay for FimA—the major pili subunit, reverse transcription quantitative PCR of fimA, and transmission electron microscopy confirmed that pili synthesis required putrescine. Comparative RNAseq analysis of a wild type and ΔspeB mutant which exhibits impaired pili synthesis showed that the latter had fewer transcripts for pili structural genes and for fimB which codes for the phase variation recombinase that orients the fim operon promoter in the ON phase, although loss of speB did not affect the promoter orientation. Results from the RNAseq analysis also suggested (a) changes in transcripts for several transcription factor genes that affect fim operon expression, (b) compensatory mechanisms for low putrescine which implies a putrescine homeostatic network, and (c) decreased transcripts of genes for oxidative energy metabolism and iron transport which a previous genetic analysis suggests may be sufficient to account for the pili defect in putrescine synthesis mutants. We conclude that pili synthesis requires putrescine and putrescine concentration is controlled by a complex homeostatic network that includes the genes of oxidative energy metabolism.

    1. Genetics and Genomics
    Yi Li, Long Gong ... Shangbang Gao
    Research Article

    Resistance to anthelmintics, particularly the macrocyclic lactone ivermectin (IVM), presents a substantial global challenge for parasite control. We found that the functional loss of an evolutionarily conserved E3 ubiquitin ligase, UBR-1, leads to IVM resistance in Caenorhabditis elegans. Multiple IVM-inhibiting activities, including viability, body size, pharyngeal pumping, and locomotion, were significantly ameliorated in various ubr-1 mutants. Interestingly, exogenous application of glutamate induces IVM resistance in wild-type animals. The sensitivity of all IVM-affected phenotypes of ubr-1 is restored by eliminating proteins associated with glutamate metabolism or signaling: GOT-1, a transaminase that converts aspartate to glutamate, and EAT-4, a vesicular glutamate transporter. We demonstrated that IVM-targeted GluCls (glutamate-gated chloride channels) are downregulated and that the IVM-mediated inhibition of serotonin-activated pharynx Ca2+ activity is diminished in ubr-1. Additionally, enhancing glutamate uptake in ubr-1 mutants through ceftriaxone completely restored their IVM sensitivity. Therefore, UBR-1 deficiency-mediated aberrant glutamate signaling leads to ivermectin resistance in C. elegans.