Biology Reference
In-Depth Information
To test titers of viral stocks:
1.
Twofold serial dilutions are used to infect cells and incubated for 6 days, with a
medium change at day 1 post-infection.
2.
At day 6, cells are harvested and further prepared according to the desired assay
and depending on the reporter gene expressed from the viral genome, such
as luciferase assay for luciferase protein or fluorescence-activated cell sorting
(FACS) assays for fluorescence markers.
For the production of DENV stocks:
1.
DENV genomic DNA is linearized with XbaI.
2.
RNA is transcribed with a T7
in vitro
polymerase kit with m7GpppA cap analog
(Promega).
3.
Transcripts are either transfected or electroporated into a variety of cell lines,
usually BHK-21 or C6/36.
4.
Virions are collected, concentrated by ultracentrifugation, and incubated with
target cells for 2 h in medium containing 2% FBS.
5.
Cells are overlaid with a reagent that prevents Brownian motion (i.e., agarose)
and assayed at the desired time post-infection by plaque assay.
10.2.6
Viral replication measurements
Various tests have been set up to examine the different stages of viral replication;
some are specific to a virus, while others might be common to the study of different
viruses. The different stages of HCV replication can generally be organized as fol-
lows: (1) viral entry, (2) fusion and uncoating, (3) translation, (4) RNA replication,
(5) viral assembly, (6) virion maturation, and (7) viral release.
In this chapter, we will describe protocols that allow us to measure the overall
efficiency of viral replication and the efficiency of individual steps of viral replica-
tion, excluding the early steps (entry, fusion, and uncoating), as no connection with
cellular LDs has yet been described.
10.2.6.1
Viral replication assays
The term viral replication refers to the entire replication cycle of viruses in cells,
from infection of a naive cell to production of new infectious virions. Assessing
if a factor plays a role in viral replication is usually the first step to undergo in a study.
This is accomplished by allowing the virus of interest to spread in the cell culture for
several replication cycles. This way, a decrease or increase in infection efficiency is
amplified at each round of replication. Such tests usually start with the infection of
cell culture at a low multiplicity of infection (MOI, lower than 0.1).
10.2.6.2
Viral transcription and translation assays
Transcription of viral RNAs only applies to viruses that use DNA as an intermediate
during genome replication (e.g., HBV). When examining the level of viral RNA, one
should keep in mind that some are used as genome replication intermediates or are
