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Determination of respiratory syncytial virus
titer from mouse lung tissues
Materials and methods
Tracy J. Ruckwardt*, Allison Malloy, and Barney S.
Graham
Vaccine Research Center, National Institute of Allergy and
Infectious Disease, National Institutes of Health, Bethesda,
Maryland, USA
* Corresponding author ([email protected])
Materials
• gentleMACS Dissociator or
gentleMACS Octo Dissociator
• gentleMACS C Tubes
• Centriguge
• Incubator (37 °C)
• 80% confluent HEp-2 cell monolayers
• 0.75% methyl cellulose
• Complete EMEM
• 10% buffered formalin
• Hematoxylin
• Eosin
Background
Infants are uniquely affected by respiratory syncytial virus
(RSV), which causes yearly winter epidemics with most
children becoming infected during their first RSV season.
Ninety percent of infants are infected by 2 years of age,
with the incidence of severe disease peaking between
6 weeks and 6 months.
Clearance of virus-infected cells depends on CD8 T cells,
and defining mechanisms of CD8 T cell regulation is
essential for understanding RSV disease pathogenesis and
guiding therapeutic interventions. CD8 T cells recognize
a virus-infected cell by detecting peptides cleaved
from viral proteins that are presented in host cell major
histocompatibility comple (MHC) molecules. The strength
of CD8 T cell responses to processed peptide epitopes
from the virus commonly assumes a predictable response
hierarchy.
Our group elucidated key differences between adult and
neonatal CD8+ T cell responses using a murine model of
RSV infection. Responses in adult hybrid CB6F1 mice were
compared to responses in neonatal CB6F1 mice with respect
to the hierarchy, function, phenotype, and clonotypic
composition of epitope specific CD8+ T cell populations.
This protocol describes our standard procedure to
determine respiratory syncytial virus titer from mouse lung
using the gentleMACS™ Dissociator.
Methods
Work fast to preserve the best viral titer. Freeze the mouse
lung tissue directly after weighing, and limit the number of
samples thawed at one time to minimize the time to plating.
Using glass vials results in the best titers because it takes
less time to freeze and thaw the supernatant.
Sample preparation
1. Transfer lung tissue to a tared glass vial containing 2 mL
of 10% EMEM.
2. Record the weight of the lung tissue for future
calculation of Log PFU/g.
3. Freeze the vial directly in a dry ice/ethanol bath and
store at - 80 °C.
Dissociation
1. Quickly thaw the sample while shaking in a 37 °C water
bath. Remove the vial before the entire supernatant is
thawed.
2. Keep thawed sample chilled. Transfer 2 mL of sample
directly into a gentleMACS C Tube.
3. Attach C Tube upside down onto the sleeve of the
gentleMACS Dissociator and run program lung_02.
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Conclusion
4. O
nce the program is finished centrifuge the sample for
15 minutes at 1000 g.
5. Proceed with plating of dissociated lung tissue.
Determination of respiratory syncytial virus titer can be
accomplished with ease using the gentleMACS Dissociator.
Plating
1. Perpare a 12-well plate containing 80% confluent
HEp-2 cell monolayers.
2. Remove the media from plate and inoculate dilutions of
clarified supernatant in triplicate in a total volume of
50 µL.
3. Rock the plate for 1 hour at room temperature, then
overlay with 1 mL of 0.75% methyl cellulose in 10%
EMEM.
4. Incubate plates for 4 days at 37 °C.
5. Fix plates with 10% buffered formalin and stain with
hematoxylin and eosin.
6. Count triplicate wells for the appropriate dilution, and
determine Log₁₀ PFU/g of tissue using the formula:
Log PFU/g tissue = Log₁₀
[(average count of triplicate wells × dilution × 40) / tissue
weight in grams]
Reference
1. R
uckwardt, T. J et al. (2011) Neonatal CD8 T-cell hierarchy
is distinct from adults and is influenced by intrinsic T
cell properties in respiratory syncytial virus infected
mice. PLoS Pathog 7(12): e1002377. doi:10.1371/journal.
ppat.1002377.
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Results
It was shown that the epitope dominance pattern of adult
mice emerges at 10 days of age and that T cells are modified
by developmental factors in an epitope-specific and
age‑dependent manner. These observations may influence
future vaccine design.
Log₁₀ PFU/g lung
6
5
4
3
2
3
5
7
9 11 13 Adult
Age at infection (days)
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V. 01
Figure 1: Viral titers in the lung at day 4 post-infection of mice infected
with RSV at the indicated day of life.