MDCK
cells
are
seeded
into
96-well
plates
at
a
density
of
1.5x105
cells/well
in
DMEM
and
incubated
at
37°C
overnight.
Cells
are
washed
2
times
with
PBS,
and
100
µL
of
DMEM
is
added
to
each
well.
DMEM
is
removed
from
all
wells
in
the
first
column
of
the
plate
and
146
µL
of
lung
homogenate
(in
DMEM)
is
added
to
these
wells
(each
biological
sample
is
added
to
4
wells).
Serial
dilutions
of
46
µL
(0.5
log
dilutions)
are
carried
out
across
the
plate.
Plates
are
incubated
at
37°C
for
1
h.
Inoculum
is
removed,
and
150
µL
of
serum-free
DMEM
with
1
µg/mL
trypsin
is
added
to
each
well.
Plates
are
incubated
at
37°C
for
3
days.
Media
is
removed
and
cells
stained
with
Crystal
Violet
solution.
Stain
is
washed
away
with
water.
Titer
is
determined
(as
defined
below
under
Definitions).
Procedure:
Lung
histopathological
analysis
Right
lung
is
removed
and
submerged
in
10%
buffered
formalin
without
inflation
for
1
wk.
Two
5-micron
thick
Hematoxylin
and
Eosin
stained
lung
sections
(separated
by
100
microns)
are
prepared
and
scored
for
a
variety
of
metrics
relating
to
the
extent
and
severity
of
immune
cell
infiltration
and
pathological
damage
[slides
were
prepared
and
blind
scored
by
the
UNC
Linberger
Comprehensive
Cancer
Center
Histopathology
Core].
Definitions
and
calculations
TCID50:
Log10
TCID100
=
2
x
Log10TCID50
=
2
x
(Xp
+
(0.5
x
D)
-
(D
x
Sp)),
where
Xp
is
the
last
dilution
where
all
replicates
of
a
given
sample
are
positive,
D
is
the
serial
dilution
log,
and
Sp
is
the
sum
of
the
proportion
of
replicates
at
all
dilutions
where
positives
are
seen
(starting
with
the
Xp
dilution)
Data
collected
by
investigator
Body
weight
*
Clinical
scores
Viral
replication
log
titers
Virus-induced
inflammation
and
damage
to
airways,
vascular
regions,
and
alveoli
(scores)
*
Immunohistochemical
(IHC)
analysis
of
viral
replication
(scores)
*
*
Additional
data
are
available,
including
data
for
mock-treated
mice
and
males
Ferris1_suppdata.xlxs.
Susceptibility
index
was
computed
by
MPD
based
on
the
summation
of
relative
weight
loss
and
clinical
score
at
day
4
post-infection.