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Inactivation of Enveloped Viruses and Killing of Cells by Fatty Acids and Monoglycerides

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Abstract and Figures

Lipids in fresh human milk do not inactivate viruses but become antiviral after storage of the milk for a few days at 4 or 23 degrees C. The appearance of antiviral activity depends on active milk lipases and correlates with the release of free fatty acids in the milk. A number of fatty acids which are normal components of milk lipids were tested against enveloped viruses, i.e., vesicular stomatitis virus, herpes simplex virus, and visna virus, and against a nonenveloped virus, poliovirus. Short-chain and long-chain saturated fatty acids had no or a very small antiviral effect at the highest concentrations tested. Medium-chain saturated and long-chain unsaturated fatty acids, on the other hand, were all highly active against the enveloped viruses, although the fatty acid concentration required for maximum viral inactivation varied by as much as 20-fold. Monoglycerides of these fatty acids were also highly antiviral, in some instances at a concentration 10 times lower than that of the free fatty acids. None of the fatty acids inactivated poliovirus. Antiviral fatty acids were found to affect the viral envelope, causing leakage and at higher concentrations, a complete disintegration of the envelope and the viral particles. They also caused disintegration of the plasma membranes of tissue culture cells resulting in cell lysis and death. The same phenomenon occurred in cell cultures incubated with stored antiviral human milk. The antimicrobial effect of human milk lipids in vitro is therefore most likely caused by disintegration of cellular and viral membranes by fatty acids. Studies are needed to establish whether human milk lipids have an antimicrobial effect in the stomach and intestines of infants and to determine what role, if any, they play in protecting infants against gastrointestinal infections.
Content may be subject to copyright.
Vol.
31,
No.
1
ANTIMICROBIAL
AGENTS
AND
CHEMOTHERAPY,
Jan.
1987,
p.
27-31
0066-4804/87/010027-05$02.00/0
Copyright
C)
1987,
American
Society
for
Microbiology
Inactivation
of
Enveloped
Viruses
and
Killing
of
Cells
by
Fatty
Acids
and
Monoglycerides
HALLDOR
THORMAR,l2*
CHARLES
E.
ISAACS,1
HANNAH
R.
BROWN,1
MARC
R.
BARSHATZKY,1
AND
TAMMY
PESSOLANO1
New
York
State
OMRDD
and
Institute
for
Basic
Research
in
Developmental
Disabilities,
Staten
Island,
New
York
10314,1
and
Institute
of
Biology,
University
of
Iceland,
Reykjavik,
Iceland2
Received
4
August
1986/Accepted
16
October
1986
Lipids
in
fresh
human
milk
do
not
inactivate
viruses
but
become
antiviral
after
storage
of
the
milk
for
a
few
days
at
4
or
23°C.
The
appearance
of
antiviral
activity
depends
on
active
milk
lipases
and
correlates
with
the
release
of
free
fatty
acids
in
the
milk.
A
number
of
fatty
acids
which
are
normal
components
of
milk
lipids
were
tested
against
enveloped
viruses,
i.e.,
vesicular
stomatitis
virus,
herpes
simplex
virus,
and
visna
virus,
and
against
a
nonenveloped
virus,
poliovirus.
Short-chain
and
long-chain
saturated
fatty
acids
had
no
or a
very
small
antiviral
effect
at
the
highest
concentrations
tested.
Medium-chain
saturated
and
long-chain
unsaturated
fatty
acids,
on
the
other
hand,
were
all
highly
active
against
the
enveloped
viruses,
although
the
fatty
acid
concentration
required
for
maximum
viral
inactivation
varied
by
as
much
as
20-fold.
Monoglycerides
of
these
fatty
acids
were
also
highly
antiviral,
in
some
instances
at
a
concentration
10
times
lower
than
that
of
the
free
fatty
acids.
None
of
the
fatty
acids
inactivated
poliovirus.
Antiviral
fatty
acids
were
found
to
affect
the
viral
envelope,
causing
leakage
and
at
higher
concentrations,
a
complete
disintegration
of
the
envelope
and
the
viral
particles.
They
also
caused
disintegration
of
the
plasma
membranes
of
tissue
culture
cells
resulting
in
cell
lysis
and
death.
The
same
phenomenon
occurred
in
cell
cultures
incubated
with
stored
antiviral
human
milk.
The
antimicrobial
effect
of
human
milk
lipids
in
vitro
is
therefore
most
liklely
caused
by
disintegration
of
cellular
and
viral
membranes
by
fatty
acids.
Studies
are
needed
to
establish
whether
human
milk
lipids
have
an
antimicrobial
effect
in
the
stomach
and
intestines
of
infants
and
to
determine
what
role,
if
any,
they
play
in
protecting
infants
against
gastrointestinal
infections.
Human
milk
contains
a
number
of
antiviral
factors
that
are
not
immunoglobulins
(5,
6,
13,
17).
Some
of
these
factors
are
located
in
the
nonlipid
fraction
of
the
milk,
but
most
studies
found
antiviral
activity
associated
with
the
lipid
fraction.
Antiviral
lipids
were
best
characterized
by
Welsh
et
al.
(21),
who
found
that
free
unsaturated
fatty
acids
and
monogly-
cerides
in
milk
inactivated
enveloped,
but
not
nonenve-
loped,
viruses.
In
a
recent
study
(9a),
we
confirmed
and
extended
the
work
of
Welsh
et
al.
We
showed
that
lipids
from
fresh
breast
milk
are
not
antiviral
but
become
active
against
enveloped
viruses
upon
storage
at
4°C
and
in
infant
stomachs,
probably
by
the
release
of
fatty
acids
from
milk
triglycerides.
In
the
present
study,
we
compared
the
effect
of
fatty
acids
and
monoglycerides
on
enveloped
viruses
and
demonstrated
that
unsaturated
fatty
acids
disrupt
both
vital
envelopes
and
cell
membranes.
MATERIALS
AND
METHODS
Cell
cultures.
Vero
cells
(African
green
monkey
kidney
cell
line;
Flow
Laboratories
Inc.,
McLean,
Va.)
were
grown
in
Eagle
basal
medium
(BME)
(GIBCO
Laboratories,
Grand
Island,
N.Y.)
with
10%
inactivated
fetal
bovine
serum
(GIBCO).
Sheep
fibroblast
cultures
were
obtained
from
the
choroid
plexus
of
a
lamb
brain
and
grown
in
15%
lamb
serum
(Colorado
Serum
Co.)
in
BME.
The
maintenance
medium
(MM)
for
Vero
cells
was
BME
with
2%
fetal
bovine
serum;
for
sheep
cells,
the
MM
was
2%
lamb
serum
in
BME.
Gentamicin
(0.1%)
was
added
to
all
media.
*
Corresponding
author.
Viruses.
Vesicular
stomatitis
virus
(VSV)
strain
Indiana
and
herpes
simplex
virus
type
1
(HSV-1)
strain
MacIntyre
were
obtained
from
the
American
Type
Culture
Collection,
Rockville,
Md.,
and
grown
in
Vero
cells.
Visna
virus
strain
K796
(19)
was
grown
in
sheep
choroid
plexus
cells.
Poliovirus
type
1
strain
Chat
was
obtained
from
R.
I.
Carp
(New
York
State
Institute
for
Basic
Research)
and
grown
in
Vero
cells.
Virus
titration.
Viruses
were
titrated
by
inoculation
of
10-fold
dilutions
(VSV,
poliovirus,
and
HSV-1
were
inocu-
kited
into
Vero
cell
cuiltures,
ahd
visna
virus
was
inoculated
into
sheep
choroid
plexus
cell
cultures)
in
96-well
microtiter
tissue
culture
plates
(Becton
Dickinson
Labware,
Oxnard,
Calif.).
A
virus
dilution
(0.1
ml)
in
MM
was
inoculated
into
each
well
with
four
wells
per
dilution.
The
plates
were
kept
for
2
to
12
days,
depending
on
the
virus,
and
examined
daily
for
cytopathic
effect.
Virus
titers
were
calculated
by
the
method
of
Reed
and
Muench
(14).
Milk
samples.
Human
milk
samples
1,
2,
and
3
were
collected
under
sterile
conditions
1
to
5
months
postpartum
and
kept
deep-frozen
at
-86°C
until
used
in
experiments.
Reagents.
Fatty
acids
and
monoglycerides
were
purchased
from
Sigma
Chemical
Co.,
St.
Louis;
Mo.
(purest
grade).
Immediately
before
use
they
were
melted
and
emulsified
in
liquid
fornm
in
BME
with
10%
fetal
bovine
serum
by
vortex-
ing
at
the
highest
speed
for
1
min.
The
emulsions
(100
mg/ml)
were
diluted
to
the
desired
concentrations
in
MM.
Enmul-
sions
of
short-chain
fatty
acids
were
neutralized
to
pH
7
by
addition
of
1
M
NaOH.
Unsaturated
fatty
acids
and
monoglycerides
were
kept
under
nitrogen,
and
emulsions
were
used
within
a
few
minutes
of
preparation.
Eserine
27
ANTIMICROB.
AGENTS
CHEMOTHER.
TABLE
1.
Free
fatty
acids
(FFA)
and
antiviral
activity
in
milka
Milk
Storage
FFA
Reduction
of
Lipoprotein
temp/time
VSV
titer
lipase
sample
(°C/days)
(mg/mi)
(log1o)
(U/ml)
1
-86/4
0.5
0
336
23/4
12.0
4.0
4/4
7.0 4.0
3
-86/4
0.5
0
20
23/4
2.0
0
4/4
2.0
0
a
The
same
results
were
obtained
for
milk
tested fresh
or
after
storage
at
-860C.
sulfate
(physostigmine;
Sigma)
and
NaCl
were
dissolved
in
water
and
diluted
in
MM
before
use
in
experiments.
Assay
of
antiviral
activity.
About
105
50%
tissue
culture
infective
doses
(TCID50s)
of
virus
were
mixed
with
a
fivefold
dilution
of
milk
in
MM
or
with
an
emulsion
of
fatty
acids
and
monoglycerides
in
MM
and
incubated
at
37°C
for
30
min.
Virus
mixed
with
MM
alone
was
used
as
a
control.
After
incubation,
the
infectivity
of
each
mixture
was
titrated
by
the
serial
dilution
endpoint
method.
Dilutions
(10-fold)
were
made
in
MM.
The
10dto
10-
dilutions
were
inoculated
into
monolayers
of
Vero
cells,
and
the
virus
titers
were
deter-
mined
as
described
above.
The
difference
between
the
titer
(log1o)
of
the
control
virus
and
the
titers
of
milk-virus
and
lipid-virus
mixtures,
i.e.,
the
reduction
of
virus
titer,
was
used
as
a
measure
of
antiviral
activity.
Preparation
of
virus
for
electron
microscopy.
VSV
was
concentrated
ahd
partially
purified
by
differential
centrifuga-
tion
in
a
Beckman
L2-65B
ultracentrifuge,
and
samples
(1010
TCID50/ml)
were
incubated
at
37°C
for
30
min
in
MM
with
or
without
emulsified
fatty
acids.
The
virus
suspensions
were
applied
to
carbon-coated
grids
and
negatively
stained
with
2%
phosphotungstic
acid,
pH
7.0.
Specimens
were
examined
by
using
a
Hitachi
HS
8-2
electron
microscope
at
50
kV.
Preparation
of
cells
for
electron
microscopy.
Monolayer
cultures
of
cells
were
incubated
for
30
min
at
37°C
either
in
MM
alone
or
with
milk
or
a
fatty
acid
emulsion.
The
cell
layers
were
then
carefully
rinsed
with
Hanks
balanced
salt
solution
and
fixed
with
2%
glutaraldehyde
in
0.1
M
cacodyl-
ate
buffer.
After
rinsing
in
buffer
and
postfixation
with
2%
osmium
tetroxide,
the
cells
were
dehydrated
through
grad-
ings
of
ethanol,
critical-point
dried,
and
sputter
coated with
10.5
nm
of
gold.
They
were
examined
in
an
ISI-SS40
scanning
electron
microscope
at
20
kV.
Estimation
of
free
fatty
acids
levels.
Lipids
from
100
,ul
of
the
milk
samples
were
extracted
with
0.5
ml
of
chloroform-
methanol
(2:1).
The
upper
phase
was
removed,
and
an
aliquot
of
the
chloroform
layer
was
separated
by
thin-layer
chromatography
on
Silica
Gel
G
(Merck
&
Co.,
Inc.,
Rahway,
N.J.)
plates
with
quantitative
standards of
oleic
acid
in
a
solvent
system
consisting
of
hexane-diethyl
ether-
acetic
acid
(70:30:1.5).
The
developed
plates
were
charred
after
spraying
with
dichromate-sulfuric
acid,
and
the
free
fatty
acids
were
quantitated
by
densitometry.
RESULTS
Relationship
between
lipolysis
and
antiviral
activity.
Previ-
ous
results
(9a)
showed
that
human
milk
becomes
active
against
enveloped
viruses
after
storage
at
4,
23,
or
-20°C
for
various
lengths
of
time.
The
antiviral
activity
is
associated
with
the
cream
fraction,
but
the
skim
fraction
is
needed
for
the
lipids
to
become
antiviral.
To
test
whether
the
appear-
ance
of
antiviral
activity
depended
on
active
milk
lipases,
we
stored
milk
samples
1,
2,
and
3
at
4°C
for
4
days
with
or
without
two
lipase
inhibitors,
5
mM
eserine
sulfate
and
1
M
NaCl
(7, 8).
The
virus
titer
(VSV)
fell
from
105
to
C101-5
TCID50
after
incubation
with
milk
stored
without
an
inhibi-
tor,
thus
showing
a
reduction
of
103
5
TCID50.
In
contrast,
virus
incubated
in
the
same
way
with
milk
which
had
been
stored
with
lipase
inhibitors
showed
no
loss
of
infectivity
at
the
concentrations
used.
The
inhibitors
had
no
effect
on
milk
which
was
already
antiviral.
Another
indication
that
the
appearance
of
antiviral
activity
in
stored
human
milk
is
associated
with
lipolysis
is
shown
in
Table
1.
Deep-frozen
human
milk
sample
1
did
not
have
a
detectable
level
of
free
fatty
acids,
but
the
level
increased
to
7
and
12
mg/ml
upon
storage
at
4
and
23°C,
respectively,
for
4
days.
Both
stored
samples
were
highly
antiviral.
The
free
fatty
acid
level
of
milk
sample
3,
oh
the
other
hand,
increased
to
only
2
mg/ml
upon
storage,
and
the
milk
did
not
become
antiviral.
Compared
with
milk
sample
3,
milk
sam-
ple
1
had
much
higher
levels
of
lipoprotein
lipase,
which
was
previously
shown
to
correlate
with
the
appearance
of
milk
antiviral
activity
(9,
9a).
Antiviral
activity
of
fatty
acids
and
monoglycerides.
A
comparison
of
the
antiviral
activity
of
a
number
of
fatty
acids
found
in
milk
(11)
is
shown
in
Table
2.
Short-chain
(butyric,
caproic,
and
caprylic)
and
long-chain
saturated
(palmitic
and
stearic)
fatty
acids
had
no
or
a
very
small
antiviral
effect
at
the
highest
concentrations
tested.
On
the
other
hand,
the
TABLE
2.
Viral
inactivation
by
incubation
with
fatty
acids
at
37°C
for
30
min
Reduction
of
virus
Fatty
acid
Concna
in
titer
(loglo)
mg/ml
(mM)
VSV
HSV-1
VVb
Butyric
(4:
0)c
10
(113)
0
NDd
ND
Caproic
(6:0)
10
(86)
0
ND
ND
Caprylic
(8:0)
10
(69)
1.8
ND
.3.2
Capric
(10:0)
4
(22)
.4.Oe
.4.0
.3.2
Lauric
(12:0)
2
(10)
-4.0
.4.0
-3.2
Myristic
(14:0)
4
(16)
.4.0
.4.0
1.7
Palmitic
(16:0)
20
(78)
1.0
1.0
0.7
Palmitoleic
(16:1)
2
(15)
-4.0
.4.0
-3.2
Stearic
(18:0)
20
(70)
0
ND ND
Oleic
(18:1
cis)
2
(7)
.4.0
.4.0
.3.2
Elaidic
(18:1
trans)
2
(7)
.4.0
ND ND
Linoleic
(18:2)
1
(3.5)
.4.0
.4.0
.3.2
Linolenic
(18:3)
1
(3.6)
.4.0
.4.0
.3.2
Arachidonic
(20:4)
0.5
(1.6)
.4.0
ND
ND
a
Concentration
of
fatty
acid
in
virus
mixtures
incubated
at
37°C
for
30
min.
All
fatty
acids
were
tested
in
a
series
of twofold
concentrations.
Shown
is
either
the
lowest
concentration
which
reduced
the
VSV
titer
by
.4.0
loglo
units
or
the
highest
concentration
tested
(butyric,
caproic,
caprylic,
palmitic,
and
stearic).
b
VV,
Visna
virus.
c
Carbon
atoms:double
bonds.
d
ND,
Not
done.
e
The
titer
(loglo)
of
the
control
virus
incubated
with
mm
was
5.5,
whereas
no
virus
was
detectable
in
the
10-2
to
10-5
dilutions
of
fatty
acid-virus
mixtures.
It
was
not
possible
to
test
these
mixtures
in
lower
dilutions
(10-1
or
undiluted)
because
they
were
toxic
to
the
cell
cultures.
Assuming
that
the
10-1
dilution
contained
infectious
virus,
the
highest
possible
titer
of
the
fatty
acid-virus
mixture
was
101-1
TCID50,
and
the
reduction
of
virus
titer
(log
1o)
would
equal
4.0
(5.5
minus
1.5).
If
the
titers
of
the
mixtures
were
less
than
10'.5,
the
reduction
of
titer
would
be
greater
than
4.0.
28
THORMAR
ET
AL.
INACTIVATION
OF
ENVELOPED
VIRUSES
BY
FATTY
ACIDS
medium-chain
saturated
and
long-chain
unsaturated
fatty
acids
were
all
antiviral
but
at
different
concentrations.
Table
2
shows
the
lowest
concentration
causing
a
>10,000-fold
reduction
in
VSV
titer.
A
2-fold-lower
concentration
either
did
not
inactivate
the
virus
or
caused
only
a
10-fold
reduc-
tion
in
titer.
Similar
results
were
obtained
for
HSV-1
and
visna
virus,
a
retrovirus.
In
contrast,
incubation
of
poliovirus
at
37°C
for
30
min
with
capric,
lauric,
myristic,
palmitoleic,
oleic,
linoleic,
linolenic,
and
arachidonic
acids,
each
at
a
concentration
of
8
mg/ml,
did
not
cause
a
signifi-
cant
reduction
of
virus
titer
compared
with
the
titer
of
poliovirus
incubated
without
fatty
acids
(104-7
TCID50).
The
sodium
salts
of
oleic
and
linoleic
acids
had
antiviral
effects
similar
to
those
of
the
free
acids.
Other
products
of
lipolysis,
e.g.,
1-monoglycerides
of
fatty
acids,
were
also
tested
for
antiviral
activity
(Table
3).
All
the
monoglycerides
tested
except
monomyristin
and
monoolein
were
antiviral
in
concentrations
5
to
10
times
lower
(mil-
limolar)
than
those
of
the
corresponding
fatty
acids.
Effect
of
fatty
acids
on
viral
particles.
To
study
the
effect
of
fatty
acids
on
virus
particles,
VSV
was
concentrated,
partly
purified,
and
then
incubated
at
37°C
for
30
min
in
MM
with
or
without
linoleic
acid.
Negative
staining
of
virus
incubated
without
fatty
acids
showed
an
abundance
of
characteristic
bullet-shaped
particles
covered
with
spikes
and
containing
coiled
nucleocapsids
(Fig.
la).
Incubation
with
0.5
mg
of
linoleic
acid
per
ml
caused
leakage
of
viral
envelopes,
allowing
the
stain
to
enter
many
particles
(Fig.
lb).
The
effect
was
far
more
pronounced
with
1
mg
of
linoleic
acid
per
ml
(Fig.
lc),
causing
particle
disintegration.
Titration
of
the
samples
used
for
electron
microscopy
showed
a
<10-fold
reduction
in
virus
titer
with
0.5
mg
of
linoleic
acid
per
ml,
whereas
1
mg/ml
caused
a
.1,000-fold
reduction.
Similar
results
were
obtained
by
negative
staining
of
VSV
incubated
with
low
concentrations
of
arachidonic
acid.
Disintegration
of
cell
membranes
by
fatty
acids.
Negative
staining
of
VSV
treated
with
fatty
acids
suggested
that
virus
inactivation
results
from
disruption
of
the
viral
envelope,
which
is
derived
from
the
host
cell
plasma
membrane.
To
study the
effect
on
cell
membranes,
monolayers
of
Vero
cells
or
sheep
fibroblasts
were
incubated
at
37°C
for
30
min
in
MM
with
or
without
1
mg
of
linoleic
acid
per
ml
and
examined
by
scanning
electron
microscopy.
Control
cells
incubated
in
MM
without
fatty
acids
showed
intact
cell
membranes
(Fig.
2c),
whereas
in
cell
layers
treated
with
1
mg
of
linoleic
acid
per
ml,
the
cell
membranes
were
partly
or
completely
TABLE
3.
Viral
inactivation
by
incubation
with
monoglycerides
at
37°C
for
30
min
Reduction
of
virus
Monoglyceride
Concna
in
titer
(log1o)
mg/mi
(MM)
VSV
HSV-1
Monocaprylin
(8:0)b
2.0
(9)
.4.0
NDc
Monocaprin
(10:0)
0.5
(2)
.4.0
.3.7
Monolaurin
(12:0)
0.25
(0.9)
.4.0
.3.7
Monomyristin
(14:0)
2.0
(13)
3.0
ND
Monoolein
(18:
1)
1.0
(2.8d)
2.3
ND
Monolinolein
(18:2)
0.25
(0.7)
.4.0
ND
a
Lowest
concentration
causing
.3.0
log1o
reduction
in
virus
titer.
b
Carbon
atoms:double
bonds.
c
ND,
Not
done.
d
Highest
antiviral
activity
of
the
concentrations
tested
(0.5
to
4
mg/ml).
The
same
results
were
obtained
when
the
monoglyceride
was
dissolved
in
ethanol
and
diluted
1:
100
in
mm
before
being
added
to
the
virus.
FIG.
1.
Negative
staining
of
VSV
particles
showing
the
effect
of
linoleic
acid.
VSV
was
incubated
at
37°C
for
30
min
in
(a)
MM,
(b)
linoleic
acid
(0.5
mg/ml
of
MM),
and
(c)
linoleic
acid
(1
mg/ml
of
MM).
(a)
Normal
intact
particles
covered
with
spikes.
(b)
Viral
envelope
no
longer
intact,
allowing
penetration
of
stain
into
most
particles.
(c)
Virus
particles
in
various
stages
of
disintegration.
Bar
=
0.1
,um.
disintegrated
(Fig.
2d),
causing
cell
lysis.
The
same
effect
was
seen
by
incubation
of
cells
with
human
milk
which
had
been
stored
at
4°C
for
4
days
(Fig.
2b).
This
milk
sample
(no.
1)
(Table
1)
contained
7
mg
of
fatty
acids
per
ml
and
was
highly
antiviral.
On
the
other
hand,
milk
sample
1
stored
at
29
VOL.
31,
1987
ANTIMICROB.
AGENTS
CHEMOTHER.
FIG.
2.
Scanning
electron
micrographs
of
cell
cultures
showing
the
effect
of
human
milk
and
linoleic
acid.
Vero
cells
were
incubated
at
37°C
for
30
min
in
(a)
human
milk,
(b)
milk
stored
at
4°C
for
4
days,
(c)
MM,
or
(d)
linoleic
acid
(1
mg/ml
of
MM).
Milk
samples
were
diluted
1:5
in
MM.
(a
and
c)
Intact
cell
membranes
with
microvilli.
(b
and
d)
Cell
membranes
partly
or
completely
disintegrated.
Bar
=
1.0
p.m.
-86°C
for
4
days
(Table
1)
showed
no
effect
on
cell
mem-
branes
(Fig.
2a).
DISCUSSION
We
have
shown
(9a)
that
human
milk
becomes
antiviral
not
only
upon
storage
but
also
in
the
stomach
of
infants
within
1
h
of
feeding.
The
appearance
of
antiviral
activity
in
stored
milk
is
related
to
the
level
of
lipoprotein
lipase
in
the
milk,
indicating
that
it
is
caused
by
the
release
of
fatty
acids
or
other
products
of
lipid
hydrolysis.
Similar
results
were
previously
reported
by
Welsh
et
al.
(20,
21).
In
this
study,
we
present
more
data
which
indicate
that
the
antiviral
effect
of
stored
human
milk
is
caused
by
lipolysis
and
show
that
of
the
nine
fatty
acids
most
commonly
found
in
human
milk
(11),
seven
are
highly
active
in
killing
enveloped
viruses.
The
polyunsaturated
long-chain
fatty
acids
were
the
most
active,
but
medium-chain
saturated
fatty
acids,
particularly
lauric
and
myristic
acids,
also
showed
activity.
Monocaprin
and
monolaurin
were
active
in
concentrations
10
times
lower
than
those
of
the
corresponding
free
acids,
but
monomyristin
was
consistently
less
active.
Long-chain
saturated
fatty
acids,
which
make
up
about
30%
of
the
fatty
acids
in
human
milk,
and
short-chain
fatty
acids,
which
are
more
common
in
cow
milk
(11),
were
not,
or
were
very
slightly,
antiviral.
The
concentrations
of
fatty
acids
found
to
reduce
viral
titers
by
.10,000-fold
in
vitro
(Table
2)
are
in
the
same
range
of
fatty
acid
concentrations
found
in
human
milk
(11).
Our
results
indicate
that
as
lipolysis
of
milk
triglycerides
proceeds,
either
during
storage
or
in
the
gastrointestinal
tract,
two
types
of
antiviral
lipids,
monoglycerides
and
free
fatty
acids,
are
produced.
It
is
possible
that
these
two
classes
of
lipid
differ
in
efficacy
against
intestinal
pathogens.
This
may
also
be
true
for
the
members
of
each
lipid
class.
Our
results
are
similar
to
those
of
earlier
studies
with
different
viruses
(6,
10,
16,
20)
and
further
establish
the
marked
antiviral
effect
of
most
fatty
acids
found
in
milk.
The
electron
microscope
study
suggests
that
the
antiviral
effect
is
caused
primarily
by
disintegration
of
viral
envelopes
by
fatty
acids.
Similar
findings
were
reported
by
Sarkar
et
al.
(17),
who
treated
mouse
mammary
tumor
virus
with
the
cream
fraction
of
human
milk
and
noted
degradation
of
the
viral
envelope.
Our
study
also
shows
disintegration
of
the
plasma
membrane
of
cultured
cells
with
concentrations
of
fatty
acids
and
stored
human
milk
that
inactivate
enveloped
viruses.
The
fatty
acids
and
monoglycerides
which
we
found
to
be
strongly
antiviral
were
shown
to
induce
fusion
of
cell
membranes
(1).
Although
the
exact
mechanism
is
not
clear,
it
has
been
suggested
that
the
fatty
acids
and
their
monoest-
ers
are
incorporated
into
the
lipid
membrane,
causing
desta-
bilization
of
the
bilayer
(3).
A
similar
mechanism
might
lead
to
the
complete
disintegration
of
cell
membranes
and
viral
30
THORMAR
ET
AL.
INACTIVATION
OF
ENVELOPED
VIRUSES
BY
FATTY
ACIDS
envelopes
we
observed.
We
did
not
compare
the
sensitivity
of
cells
and
enveloped
viruses
at
various
fatty
acid
concen-
trations.
However,
a
study
using
hydrophobic
alcohols
showed
that
viruses
are
killed
at
concentrations
that
appar-
ently
had
no
effect
on
cultured
cells
(18).
Several
studies
have
indicated
a
lower
incidence
of
infec-
tions,
particularly
gastrointestinal,
in
breast-fed
versus
bot-
tle-fed
infants
(4,
12).
However,
the
role
of
milk
fatty
acids
and
their
derivatives
in
protecting
babies
against
illness
is
not
established,
despite
their
well-known
antimicrobial
ef-
fect
in
vitro.
Although
most
known
gastrointestinal
viruses
are
nonenveloped,
necrotizing
enterocolitis
in
infants
is
caused
by
an
enveloped
virus,
i.e.,
a
human
enteric
coronavirus
(15).
Giardia
lamblia,
an
intestinal
protozoan
parasite
infecting
children,
is
killed
by
milk
fatty
acids
in
vitro
(9),
suggesting
the
possibility
of
a
giardiacidal
effect
of
fatty
acids
in
the
intestines.
Since
fatty
acids
lyse
cells
by
disrupting
their
plasma
membranes,
it
is
likely
that
they
kill
not
only
giardia
but
also
other
parasitic
protozoa.
Although
a
few
studies
have
demonstrated
antimicrobial
activity
of
human
and
animal
stomach
contents
after
milk
feeding
(2,
9a),
much
more
work
is
needed
to
characterize
the
active
factors
and
to
establish
their
role
in
prevention
of,
and
recovery
from,
gastrointestinal
infections.
ACKNOWLEDGMENTS
We
thank
R.
Pullarkat
for
quantitation
of
milk
fatty
acids,
R.
Weed
for
photographic
supervision,
and
E.
M.
Riedel
for
typing
the
manuscript.
LITERATURE
CITED
1.
Ahkong,
Q.
F.,
D.
Fisher,
W.
Tampion,
and
J.
A.
Lucy.
1973.
The
fusion
of
erythrocytes
by
fatty
acids,
esters,
retinol
and
alpha-tocopherol.
Biochem.
J.
136:147-155.
2.
Canas-Rodriguez,
A.,
and
H.
Williams
Smith.
1966.
The
identi-
fication
of
the
antimicrobial
factors
of
the
stomach
contents
of
sucking
rabbits.
Biochem.
J.
100:79-82.
3.
Cullis,
P.
R.,
and
M.
J.
Hope.
1978.
Effects
of
fusogenic
agent
on
membrane
structure
of
erythrocyte
ghosts
and
the
mecha-
nism
of
membrane
fusion.
Nature
(London)
271:672-674.
4.
Cunningham,
A.
S.
1979.
Morbidity
in
breast-fed
and
artifically
fed
infants.
J.
Pediatr.
95:685-689.
5.
Falkler,
W.
A.,
Jr.,
A.
R.
Diwan,
and
S.
B.
Halstead.
1975.
A
lipid
inhibitor
of
dengue
virus
in
human
colostrum
and
milk;
with
a
note
on
the
absence
of
anti-dengue
secretory
antibody.
Arch.
Virol.
47:3-10.
6.
Fieldsteel,
A.
H.
1974.
Non-specific
antiviral
substances
in
human
milk
active
against
arbovirus
and
murine
leukemia
virus.
Cancer
Res.
34:712-715.
7.
Fredrikzon,
B.,
0.
Hernell,
L.
Blackberg,
and
T.
Olivecrona.
1978.
Bile
salt-stimulated
lipase
in
human
milk:
evidence
of
activity
in
vivo
and
of
a
role
in
the
digestion
of
milk
retinol
esters.
Pediatr.
Res.
12:1048-1052.
8.
Hernell,
O.,
and
T.
Olivecrona.
1974.
Human
milk
lipases.
I.
Serum-stimulated
lipase.
J.
Lipid
Res.
15:367-374.
9.
Hernell,
O.,
H.
Ward,
L.
Blackberg,
and
M.
E.
A.
Pereira.
1986.
Killing
of
Giardia
lamblia
by
human
milk
lipases:
an
effect
mediated
by
lipolysis
of
milk
lipids.
J.
Infect.
Dis.
153:715-720.
9a.Isaacs,
C.
E.,
H.
Thormar,
and
T.
Pessolano.
1986.
Membrane
disruptive
effect
of
human
milk:
inactivation
of
enveloped
viruses.
J.
Infect.
Dis.
154:966-971.
10.
Kabara,
J.
J.
1980.
Lipids
as
host-resistance
factors
of
human
milk.
Nutr.
Rev.
38:65-73.
11.
Lammi-Keefe,
C.
J.,
and
R.
G.
Jensen.
1984.
Lipids
in
human
milk:
a
review.
II.
Composition
and
fat-soluble
vitamins.
J.
Pediatr.
Gastroenterol.
Nutr.
3:172-198.
12.
Larsen,
S.
A.,
Jr.,
and
D. R.
Homer.
1978.
Relation
of
breast
versus
bottle
feeding
to
hospitalization
for
gastroenteritis
in
a
middle-class
U.S.
population.
J.
Pediatr.
92:417-418.
13.
Matthews,
T.
H.
J.,
C.
D.
G.
Nair,
M.
K.
Lawrence,
and
D.
A.
J.
Tyrrell.
1976.
Antiviral
activity
in
milk
of
possible
clinical
importance.
Lancet
ii:1387-1389.
14.
Reed,
L.
J.,
and
M.
Muench.
1938.
A
simple
method
of
estimating
50
per
cent
end
points.
Am.
J.
Hyg.
27:493-497.
15.
Resta,
S.,
J.
P.
Luby,
C.
R.
Rosenfeld,
and
J.
D.
Siegel.
1985.
Isolation
and
propagation
of
a
human
enteric
coronavirus.
Science
229:978-981.
16.
Sands,
J.
A.,
D.
A.
Auperin,
P.
D.
Landin,
A.
Reinhardt,
and
S.
P.
Cadden.
1978.
Antiviral
effect
of
fatty
acids
and
deriva-
tives:
lipid-containing
bacteriophages
as
a
model
system,
p.
75-95. In
J.
J.
Kabara
(ed.),
The
pharmacological
effect
of
lipids.
The
American
Oil
Chemists
Society,
Champaign,
Ill.
17.
Sarkar,
N.
H.,
J.
Charney,
A.
S.
Dion,
and
D.
H.
Moore.
1973.
Effect
of
human
milk
on
the
mouse
mammary
tumor
virus.
Cancer
Res.
33:626-629.
18.
Snipes,
W.,
and
A.
Keith.
1978.
Hydrophobic
alcohols
and
di-tert-butyl
phenols
as
antiviral
agents,
p.
63-73.
In
J.
J.
Kabara
(ed.),
The
pharmacological
effect
of
lipids.
The
Ameri-
can
Oil
Chemists
Society,
Champaign,
Ill.
19.
Thormar,
H.,
M.
R.
Barshatzky,
K.
Arnesen,
and
P.
Kozlowski.
1983.
The
emergence
of
antigenic
variants
is
a
rare
event
in
long-term
visna
virus
infection
in
vivo.
J.
Gen.
Virol.
64:1427-1432.
20.
Welsh,
J.
K.,
M.
Arsenakis,
R.
J.
Coelen,
and
J.
T.
May.
1979.
Effect
of
antiviral
lipids,
heat,
and
freezing
on
the
activity
of
viruses
in
human
milk.
J.
Infect.
Dis.
140:322-328.
21.
Welsh,
J.
K.,
I.
J.
Skurrie,
and
J.
T.
May.
1978.
Use
of
Semliki
forest
virus
to
identify
lipid-mediated
antiviral
activity
and
anti-alphavirus
immunoglobulin
A
in
human
milk.
Infect.
Im-
mun.
19:395-401.
VOL
.
31,
1987
31
... Although the exact mechanism of how MCFAs inactivate viruses is unknown, there seems to be a consensus that the fatty acids cause the disintegration of the bilayer lipid envelope (Kohn et al., 1980b;Thormar et al., 1987). Medium-carbon saturated fatty acids penetrate the viral envelope via hydrophobic effect, facilitating it permeable to small molecules and thereby inactivating the virus. ...
... Among many feed additives that examined the efficacy of curbing viral transmission in swine feed, medium-chain fatty acids (MCFAs), a group of fatty acids with 6 to 12 carbon atoms, have emerged as a promising mitigant due to their antiviral properties. MCFAs could penetrate the viral membrane via a hydrophobic effect causing disintegration of the lipid bilayer and thereby inactivating the viruses by dissolving their envelopes (Kohn et al., 1980a(Kohn et al., , 1980bThormar et al., 1987). Coconut oil (CO) and palm kernel oil (PKO) are the best-known natural sources of medium-chain saturated fatty acids, including caprylic (C8), capric (C10), and lauric acid (C12), accounting for more than 50% of the fatty acids in these two oils . ...
... & 2016). Although the precise mechanism of how MCFAs inactivate viruses remains unknown, there seems to be a consensus that the fatty acids cause the disintegration of the bilayer lipid envelope(Thormar et al., 1987;Kohn et al., 1980b). It is hypothesized that MCFA could penetrate the viral envelope via a hydrophobic effect, facilitating it permeable to small molecules and thereby preventing virus attachment to host cells and, ultimately, inhibiting viral replication. ...
Article
The objective of the project were to: 1) evaluate the feeding value of proso millet as a potential substitute for corn in corn-soybean meal-based diets for pigs; and 2) investigate the relationship between feed ingredients and virus survivability, followed by examining feed additive candidates that could mitigate the risk of transmission of viral pathogens. In Exp. 1, 36 barrows were fed 1 of 4 dietary treatments, including: Diet 1, corn-soybean meal-based (control), and Diets 2, 3, and 4 had proso millet replacing 33%, 67%, and 100% of corn in the control diet, respectively. During Phase 3, pigs consuming the proso millet diets (Diets 2, 3, and 4) had greater ADFI than pigs consuming the basal diet, but ADG:ADFI was not different among groups. The growth parameters (i.e., ADG, ADFI, and ADG:ADFI) were not affected by treatments during the whole experimental period (from Phase 1 to Phase 4), suggesting that the level of corn replacement up to 100% by proso millet did not affect pig growth. In Exp. 2, ground corn, SBM, DDGS, and a complete feed, were inoculated with PRRSV, followed by a cell-based antiviral assay and qRT-PCR to evaluate the infectivity of each virus-inoculated ingredient. A significant reduction of infectious PRRSV was observed in ground corn, DDGS and complete feed, while SBM retained the highest amount of the infectious virus among ingredients and controls. The results suggested that the survivability of PRRSV in feed appears to be influenced by ingredient type. The follow-up experiment was conducted to assess the antiviral activity of selected MCFAs, as determined by antiviral assay and qRT-PCR. The antiviral assay showed that all tested MCFA and MCFA blends inhibit PRRSV in a dose-dependent manner, indicating that MCFAs and the specific mixture of fatty acids may potentially serve as mitigants for reducing the risk of PRRSV transmission via feed. Advisor: Hiep L. X. Vu & Phillip S. Miller
... At a concentration above 750 μg mL -1 , it was found that lauric acid suppressed the titer of the vesicular stomatitis virus (VSV) by 98%. Similarly, H. Thormar et al. [60] found that lauric acid decreased the growth of VSV and herpes simplex virus (HSV) at a concentration of 2 mg mL -1 with a reduced value of virus titer (log 10 ) of > 4 and 3.2 for HSV and visna virus, respectively [60]. In a separate report, Isaacs et al. [62] investigated the antiviral activity against VSV in human and bovine milk. ...
... At a concentration above 750 μg mL -1 , it was found that lauric acid suppressed the titer of the vesicular stomatitis virus (VSV) by 98%. Similarly, H. Thormar et al. [60] found that lauric acid decreased the growth of VSV and herpes simplex virus (HSV) at a concentration of 2 mg mL -1 with a reduced value of virus titer (log 10 ) of > 4 and 3.2 for HSV and visna virus, respectively [60]. In a separate report, Isaacs et al. [62] investigated the antiviral activity against VSV in human and bovine milk. ...
... Vesicular stomatitis virus (VSV) The infectivity of cell with VSV was titrated by the serial dilution endpoint method [60] Lauric acid is extremely potent to destroy VSV Vesicular stomatitis virus (VSV) Plaque assay in Linbro plates [61] Lauric acid leakages the host cell membrane of M protein ...
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Virgin coconut oil is obtained by wet processing of coconut milk using fermentation, centrifugation, enzymatic extraction, and the microwave heating method. Presently, VCO has several positive effects and benefits to human health, hence, it is regularly consumed and widely known as a unique functional food. VCO contains lauric acid (45 to 52 %). By lipase in the digestive system, VCO can undergo a breakdown into lauric acid, 1‐monolaurin, and 2‐monolaurin. These components have both hydrophilic and lipophilic groups and are also recognized as excellent antimicrobial lipids. Furthermore, lauric acid and monolaurin can be used as antibacterial, antifungal, and antiviral with broad‐spectrum inhibition. Lauric acid and monolaurin have a strong ability to destroy gram‐positive bacteria, especially S. aureus, fungi such as C. Albicans, and viruses including vesicular stomatitis virus (VSV), herpes simplex virus (HSV), and visna virus (VV). Lauric acid and monolaurin interact with certain functional groups located in the cell membrane and can cause damage to the cell. In general, the potential of VCO as healthy food is contributed by lauric acid and monolaurin which are antimicrobial agents. Virgin coconut oil (VCO) is a functional edible oil, rich with lauric acid. VCO can be converted into partial lipids, i.e., lauric acid, 1‐monolaurin, and 2‐monolaurin by lipase in the digestive system. These compounds are known as antimicrobial lipids based on their excellent activity in inhibiting the growth of broad‐spectrum microbial. The inhibition mechanism of lauric acid and monolaurin as antibacterial, antifungal, and antiviral agents is also discussed
... GC-MS analysis of methanol extract showed 9,12-octadecadienoic (Z,Z)-, dodecanal and n-butyric acid 2-ethylhexyl ester. According to a study conducted by Hayashi et al. (1995) and Abubakar and Majinda (2016), the compound 9,12-octadecadienoic (Z,Z)-has best antibacterial while n-butyric acid 2-ethylhexyl ester and dodecanal have good antiviral activity (Thormar et al. 1987;Hayashi et al. 1995). ...
... The bioactive compound trans-p-mentha-1(7),8-dien-2-ol and octanal was considered best antibacterial agent from the work conducted by Reichling et al. (2009) andSpencer et al. (2021) on different amphibian oils. The antiviral activity of 1-Butanol extract was due to presence of n-butyric acid 2-ethylhexyl ester, octanal, dodecanal and 2,4-decadienals reported from earlier research work showed by Thormar et al. (1987), Hayashi et al. (1995), Reichling et al. (2009), andSetzer (2016). ...
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There is an urgent need to develop natural antimicrobials for the control of rapidly mutating drug-resistant bacteria and poul- try viruses. Five extracts were prepared using diethyl ether, ethyl acetate, methanol, 1-butanol and n-hexane from abdominal fats of Varanus griseus locally known as Indian desert monitor. Antibacterial, antioxidant and antiviral activities from oil extracts were done through disc difusion method, stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging assay and in ovo antiviral assay, respectively. The gas chromatography mass spectrometry (GC–MS) analyses were used to determine principal active compounds and chemical profle of each oil extract. n-Hexane extract showed clear zones of inhibition (ZOI) against Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae (12±0.5 mm, 9±0.5 mm, and 9±0.5 mm) while diethyl ether extract exhibited signifcant antibacterial activity (11±0.5 mm) against Proteus vulgaris only. In case of drug-resistant strains, methanol extract was active (6±0.5 mm) against Staphylococcus aureus, whereas n-hexane extract has shown ZOI 11±0.5 mm against P. aeruginosa. Range of percentage scavenging activity of V. griseus oil extracts from DPPH free radical assay was 34.9–70.7%. For antiviral potential, growth of new castle disease virus (NDV) was efectively inhibited by all fve extracts (HA titer=0–4). The highest antiviral activity against avian infuenza virus (H9N2) was observed from methanol, diethyl ether and 1-Butanol oil extracts with HA titers of 2, 2 and 0, respectively. Methanol, diethyl ether, 1-butanol and n-hexane oil extracts produced best hemagglutination assay (HA) titer values (0, 0, 4 and 0) against infectious bronchitis virus (IBV). Ethyl acetate and 1-Butanol extract exhibited good antiviral potential against infec- tious bursal disease virus (IBDV) with indirect hemagglutination assay (IHA) titers of 8 and 4, respectively. Main classes of identifed compounds through gas chromatography were aldehydes, fatty acids, phenols and esters. GC–MS identifed 11 bioactive compounds in V. griseus oil extracts. It is summarized that V. griseus oil has strong antioxidant activity and good antimicrobial potential because of its bioactive compounds.
... Los ácidos grasos polinsaturados de cadena larga muestran mayor actividad antiviral [13], [14], tales como el ácido araquidónico. Sin embargo, los ácidos grasos de cadena media también muestran buena actividad en contra de los virus, particularmente el ácido láurico y el mirístico [14]. ...
... Los ácidos grasos polinsaturados de cadena larga muestran mayor actividad antiviral [13], [14], tales como el ácido araquidónico. Sin embargo, los ácidos grasos de cadena media también muestran buena actividad en contra de los virus, particularmente el ácido láurico y el mirístico [14]. Por esto, es que se tienen en cuenta aquellos aceites vegetales comestibles con los mayores contenidos de estos ácidos grasos. ...
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Las enfermedades respiratorias virales, como el nuevo coronavirus (2019-nCoV), el cual se ha extendido a muchos otros países alrededor del mundo, causan muertes, problemas económicos y sociales. En el presente artículo se estudian y abordan compuestos no convencionales que actúan como agentes de protección y desinfección eficaces en contra del 2019-nCoV. Los compuestos seleccionados se basan en la capacidad de destruir las proteínas estructurales de este virus e inhibir sus mecanismos de propagación e invasión de las células sanas. Por lo tanto, varios aceites vegetales y comestibles fueron propuestos, de acuerdo con su capacidad de disolución de las proteínas (GP120), su tensión superficial y su composición de ácidos grasos.