Biology Reference
In-Depth Information
with a luciferase reporter gene cloned in its genome. More details about titration
methods are provided in the next paragraph.
10
6
cells are electroporated with viral RNA and plated into six-well
plates (two to three plates per electroporation).
2.
On day 2, cells are washed with PBS and fresh medium is added (1-2 ml per
well). Drugs can be added here. Naive cells are plated in 12- or 24-well plates
(three wells per transfected point to perform the infections in triplicate).
3.
On day 3, supernatants are collected, filtered with 0.45
m
m filters (individually),
and used to infect naive cells (infections are done in triplicate with 0.3-0.6 ml).
An aliquot of each supernatant is kept and diluted in series to be used for ELISA.
For details about the titration of the supernatant, see
Section 2.6.6
.
4.
To isolate the intracellular viral particles, “producing cells” are washed and lysed
by three freeze-thaw cycles and then centrifuged to remove the cell debris.
The intracellular viruses can be titrated similarly to the secreted ones
(see
Section 2.6.6
).
5.
To measure the amount of viral particles (infectious or not) produced from
serum samples, one can use an ELISA that targets the viral antigen used in the
clinical diagnosis of viral infection.
1.
On day 1, 4
Western blot analysis of the cell lysate should be done to check efficient translation
and processing of viral proteins. Luciferase measurements can also be performed if
the virus contains a reporter gene to quantify the viral translation.
10.2.6.6
Viral titration assays
After isolation of viral particles produced under the desired conditions (intracellular
or released), the infectivity of the viral particles is often quantified. The presence of
reporter genes greatly facilitates this process. Here, we will describe a protocol for a
viral construct without reporter and then provide protocols that can be used with
luciferase and fluorescent reporter genes.
1.
The naive cells are plated 1 day before infection.
2.
Viruses from producing cells are isolated, filtrated, and used to infect naive cells
in triplicate. Depending on the efficacy of viral production, dilution of the viral
samples can be done. This is usually not required for HCV samples.
3.
Infection is done for 3 h at 37
C. Afterwards, the cells are washed once with
PBS, and fresh medium is added.
4.
At 24-48 h after infection, the number of infected cells, reflecting the number
of infectious particles produced upstream, is quantified. When no reporter
gene is present in the viral construct, the best method is to stain the infected
cells with a specific antibody against one of the viral antigens and measure
the percentage of infected cells by FACS. A similar analysis can be
performed by microscopy but tends to be more tedious.
When the viral construct used contains a fluorescent reporter gene, such as GFP or
mCherry, the cells can be analyzed by FACS right after fixation (which is not
