Investigating the Human Intestinal Virome, 2020, Hsieh (Ph.D. Thesis)

Discussion in 'ME/CFS research' started by Dolphin, May 10, 2022.

  1. Dolphin

    Dolphin Senior Member (Voting Rights)

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    Only a small part of this appears to be on ME/CFS

    From: Dr. Marc-Alexander Fluks


    Source: University of East Anglia
    Date: December 2020
    URL: https://ueaeprints.uea.ac.uk/id/eprint/81750/

    https://ueaeprints.uea.ac.uk/id/eprint/81750/1/2020HsiehS-YPhD.pdf


    Investigating the human intestinal virome
    -----------------------------------------
    Shen-Yuan Hsieh
    - Quadram Institute Biosciences, Faculty of Medicine and Health
    Sciences, Norwich Medical School. University of East Anglia, U.K.


    Abstract

    The human intestinal virobiota consists of highly complex and diverse
    viruses and virus-associated genes (termed the 'virome'), dominated by
    bacteriophages. Numerically, viruses have been considered the most
    abundant and diverse biological entities on Earth, estimated to be
    approximately 1031 in number. In the human gastrointestinal tract (GIT),
    virus-to-microbe ratio (VMR) may be close to 1:1, while it may reach
    20:1 at mucosal surfaces and within the mucus layer, in total numbering
    1010-1015 virus-like particles (VLPs). Recent studies suggested that
    changes in the intestinal virome may lead to chronic GI-inflammation and
    intestinal microbial dysbiosis, thereby triggering diseases such as
    myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). Thus, this
    thesis aimed to develop robust and reliable protocols for characterising
    the human faecal microbiome that can be applied to analysing the virome
    in patients with severe ME/CFS.

    This thesis first aimed to develop a reliable and reproducible protocol
    for VLP isolation from human faeces and for VLP quantification using a
    digital image analysis (DIA)-based method. Protocols were then optimised
    for VLP DNA extraction to obtain DNA of sufficient quality and quantity
    for next generation sequencing (NGS). As part of these studies, I
    developed a bioinformatics pipeline for viral metagenomic analysis, and
    also, I determined the extent of PCR amplification bias in
    virome-enriched, uncultivated virus genomes (UViGs) by comparing the
    methods of linker amplified shotgun library (LASL) and non-amplified
    shotgun library (NASL) preparations. The optimised protocols and
    bioinformatics pipeline were then applied to the initial analysis of the
    faecal virome of severely affected ME/CFS patients and same household
    healthy control individuals (SHHC).

    The optimised protocol is comprised of (1) homogenisation of faecal
    samples by vortexing without the use of bead-beating, followed by
    incubation on ice to facilitate the release of VLPs from solid
    materials; (2) partition of crude faecal matter, dietary debris and
    virions/VLPs by two-round of high-speed centrifugation; (3) sequential
    filtration using 0.8 mum and 0.45 mum filter; (4) PEG precipitation; (5)
    DNase and RNase treatment; (6) proteinase K digestion; (7) viral capsid
    lysis with SDS lysis buffer; (8) Phenol/Chloroform/Isoamyl alcohol
    extraction; (9) DNA purification using silica-based spin columns, and
    (10) DNA concentration using a vacuum-based condenser. Using three
    independent stool samples to evaluate reproducibility, VLP DNA yields
    were between 67.2 ng and 94.8 ng per gram of faeces. For VLP
    quantification, manual counting-based DIA method was more accurate and
    reliable than automated counting-based method.

    Using an optimised bioinformatics pipeline to analyse UViGs from PCR and
    non-PCR virome-derived datasets, I found that misrepresentation of
    certain viruses may occur after amplification in their relative
    abundance. In alpha diversity, the UViGs from non-PCR datasets generally
    have higher richness and diversity than those from PCR datasets,
    suggesting that PCR is likely to lower viral richness and diversity.
    Moreover, the major differences in beta diversity were more likely to be
    driven by a high level of intestinal virome individuality between
    donors, while amplification bias may have a minor effect on the beta
    diversity of viruses in the PCR datasets. In addition, in an initial
    analysis of comparing UViG similarity networks, I found that there is no
    significant difference between both datasets but further investigation
    is required.

    In comparing faecal samples from ME/CFS and SHHC, VLPs with nucleic
    acid-containing capsids in SHHC samples were higher than those of severe
    ME/CFS patients, although the variation and diversity of VLP were seen
    in both sets of faecal samples. Transmission electron microscopy (TEM)
    analysis identified Siphoviridae as the most prominent virus in both
    ME/CFS patients and SHHC VLP samples. Moreover, giant Siphoviruses were
    occasionally detected, suggesting potential novel strains are present in
    these samples. The biological meaning of these findings is not clear and
    requires further investigation. In ongoing work, the optimised protocol
    and bioinformatic pipeline is being applied to investigating the
    composition of the intestinal virome in severe ME/CFS patients and SHHC.

    --------
    (c) 2020 University of East Anglia
     
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  2. SNT Gatchaman

    SNT Gatchaman Senior Member (Voting Rights)

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