Plaque Essay

VIRAL QUANTIFICATION USING A PLAQUE ASSAY
Doctors prescribing antivirals for chronic infections need to continuously monitor the severity of the infection. They do this by determining the concentration of viral particles in a given sample. This    value    is    often    referred    to    as    the    viral    load,    the    viral    burden,    or    the viral titer. Knowing their viral load helps patients better manage their condition and make decisions about activities that could potentially spread an active virus. 

There are many methods for measuring viral load. One highly accurate    method    is    to    measure    the    number    of    specific    viral    DNA    or    RNA    sequences    in    a    sample    using    either    quantitative    PCR    or    reverse transcriptase PCR. This method requires access to both advanced equipment and highly trained technicians. Another popular    tool    is    the    Enzyme-linked    Immunosorbent    Assay    (ELISA),    which    uses    antibody-antigen    binding    to    detect    specific    antiviral    proteins in a patient's sample. ELISAs are quick and sensitive but are    only    indirect    measures    of    viral    load.    Finally,    a    plaque    assay    can    be    used    to    determine    viral    load    based    on    the    number    of    cells infected by active viruses in a patient’s sample. 

During    a    plaque    assay,    the    potentially    infected    sample    is    serially    diluted    and    inoculated    into    a    host    cell    culture.    This    is    followed    by    an    incubation    period    that    allows    any    virions    present    to    attach    to    these    new    cells.  Next,    a    layer    of    agar    is    poured over the potentially infected cells. Because the host cell culture is a single layer of cells and because the agar blocks the    virus    from    moving    vertically    the    only    way    for    a    virus    to    spread    is    by    infecting    a    neighboring    cell.  Consequently,    during    the    next    incubation    period,    a    circular    zone    of    infection    will    form    around    any    cell    that    was    infected.  This    circular    zone    is    known    as    a    plaque    and    can,    in    fact,    be    visualized    by    the    naked    eye    –    particularly    when    additional    dyes    that    emphasize    the    difference    between    uninfected,    living    cells    and    infected,    dead    cells    are    used.    

During    a    plaque    assay,    the    viral    load    is    calculated    as    a    measure    of    plaque    forming    unit    (PFU)    per    milliliter.    To    account    for    variation    and    errors    in    counting,    only    plates    containing    between    10    and    100    plaques    are    used    to    calculated    PFU    measurements.    In    addition,    multiple    plates    are    prepared    per    sample    because    the    viral    concentration    in    a    patient    can    vary    tremendously    over    time.  For    example,    someone    who    has    recently    been    infected    with    HIV    can    have    over    40    million    viral    copies per mL of blood while someone on treatment with an undetectable viral load can have less than 50 viral copies per mL.    To    account    for    this    range    scientists    will    prepare    a    serial    dilution    of    the    initial    sample,    create    a    plate    for    each    dilution,    and    then incorporate the dilution strength into their PFU calculations. 

Viral Quantification Steps for Lab
Summary - Dengue Defense Project
The Dengue Defense Project studies the effectiveness of two potential anti-dengue fever medications that have recently been approved for testing in humans. Measurement of viral load, using a laboratory technique called a plaque assay, will be used to evaluate the effectiveness of each medication over time. Plaque assays monitor the movement of viruses into cells on agar plates in the lab.

Take detailed notes on each step of the Plaque Essay

• Measuring Viral Load

• Labeling Plates

• Infected Cells

• Incubate

• Plaques

• Counting Plaques

For the viral quantification experiment, blood samples were taken from two patients newly infected with dengue fever, before and during treatment with an antiviral medication. You are responsible for preparing four plaque assay plates at different concentrations to determine the viral load of one patient at one time point. At the lab, you will pool data with other teams to share results and evaluate the effectiveness of each treatment.