FEMS MicrobiologyLetters 29 (1985) 207-210
Published by Elsevier
207
FEM 02198
Sensitivity of influenza A viruses to human interferons in human
diploid cells
(Influenza A virus; human interferon; recombinant interferon; plaque assay)
M.A. H o r i s b e r g e r a n d K. de Staritzky
Division Pharma, Bioteehnology, Ciba-Geigy Ltd, CH-4002 Basle, Switzerland
Received 20 March 1985
Revision received31 May 1985
Accepted 3 June 1985
1. SUMMARY
The sensitivity of influenza virus to the action
of natural human interferon (IFN)-a +/3 and -3',
and to the action of highly purified recombinant
HulFN-aB, -aD, and -aF, has been investigated.
A plaque assay for the fowl-plague strain of influenza A virus has been established using human
embryonic foreskin (HEF) cells. The sensitivity of
influenza virus to all IFNs tested in this assay was
comparable to that shown by vesicular stomatitis
virus (VSV) which was taken as the reference
standard. The high sensitivity to IFN action found
for the fowl-plague strain was confirmed for the
WSN strain of human origin in a yield reduction
assay.
2. I N T R O D U C T I O N
Viruses of different classes vary widely in their
sensitivity to IFN [1]. The degree of sensitivity of
influenza A viruses to human IFNs in human cells
has not been previously studied, although it is
known that treatment of human cells with human
IFNs-a and -fl inhibits somewhat the replication
of influenza virus [2,3]. No data are available on
the anti-influenza virus action of human IFN-3,.
This lack of knowledge is probably due to the
difficulty of growing and plaquing influenza viruses
in human cells.
The object of the present study was to compare
the level of sensitivity of influenza virus to human
IFNs in a human cell system with that of a reference virus, VSV. This virus replicates in many
different cell species and is widely used for detecting and standardizing antiviral activities of IFN.
For these comparative studies we have established
a plaque assay for influenza virus in human cells.
The result of a plaque reduction assay is independent of the m.o.i, which otherwise affects the
apparent degree of protection induced by a fixed
concentration of IFN in a yield-reduction test [4].
The plaque test also facilitates the comparison of
two different viruses when their sensitivity to an
inhibitor has to be determined. Moreover, a 50%
reduction in plaque formation is measurable with
precision.
3. M E T H O D S
3.1. Cells and viruses
H E F cells (Flow 7000) were propagated in
0378-1097/85/$03.30 © 1985 Federation of European MicrobiologicalSocieties
208
minimal Eagle's medium containing 10% fetal calf
serum (FCS). H E F cells were subcultured using a
solution of trypsin (1 : 250) and EDTA (GIBCO).
Working stocks of VSV (Indiana) and of influenza A viruses, strain fowl plague ( R o s t o c k /
3 4 / H 7 N 1 ) and WSN (H1N1) were prepared from
the allantoic cavity of 10-day-old embryonated
eggs. Viruses were not passaged on human cells.
Viruses were titered by plaque formation on
primary calf-kidney cultures.
3.2. Plaque test
Cell cultures were incubated in the presence of
serial dilutions of IFN for 18 h before virus challenge. Cultures (6 cm in diameter) were infected
with 40-80 p.f.u, of virus diluted in Hanks' balanced salt solution containing 0.1% of bovine
serum albumin, for 60 min at 20°C. Cultures were
then washed and a layer consisting of MEMN a H C O 3 containing 5% FCS and 0.7% Agarose A
(Pharmacia Fine Chemicals) was poured onto the
cultures. Plaques were counted first directly and
then after staining with neutral red or crystal
violet.
3.3. Interferons
Human leukocyte reference IFN was N.I.H.
G-023-902-527. Human IFN-a + fl (a mixture of
a and/3 IFNs) induced by Newcastle disease virus
in Namalwa cells, was partially purified to a
specific activity of 3 x 105 international units
( I U ) / m g of protein measured on bovine cells with
VSV. Highly purified ( > 95%pure) preparations of
recombinant human leukocyte IFN-aB, -aD, and
-aF had specific activities of 1.6 x 108, 1.9 x 108,
and 1.1 x 108 I U / m g , respectively, as measured
by plaque reduction of VSV on primary calf-kidney cultures. Human IFN-y induced by concanavalin A in human mononuclear cells from
buffy coats was titered on WISH cells challenged
with Mengo virus. The units of IFN-y were standardized with the human leukocyte reference interferon N.I.H. G-023-902-527 in order to perform
all comparative studies with equivalent IFN concentrations. The preparation of IFN-y was essentially free of IFN-a +/3 as shown by its complete
inactivation at pH 2. The preparation had no
measurable IFN-activity on bovine cells indicating
that a putative contamination with IFN-a represented less than 0.1% of total IFN activity.
4. RESULTS
The fowl-plague strain of influenza A virus was
found to plaque on H E F diploid cells (Flow 7000).
The HEF cells were infected 40-48 h after subpassaging and 18 h after the addition of serial
dilutions of IFN. Plaques were counted 48 h after
infection. The plaque formation did not necessitate the incorporation of trypsin in the agarose
overlay [5]. Addition of trypsin did not increase
significantly the number of plaques (not shown).
The action of IFN reproducibly reduced the
number of plaques, usually without affecting their
size (Fig. 1). Plaque formation by FPV was sensitive to the action of natural IFN-c~ + fl and of
natural IFN-~, (Fig. 2). The inhibition curves for
influenza virus and VSV paralleled each other,
thus demonstrating that both viruses displayed
Fig. 1. Plaques formed by the fowl-plague strain of influenza A
virus on h u m a n embryonic foreskin cells. Culture (a) was left
untreated and cultures (b)-(d) were treated with 2.4, 8 and 24
p g / m l of recombinant I F N - a F for 18 h before virus infection.
Plaques developed in 48 h and they were stained with crystal
violet.
209
100
IFN-O(÷/~ Namalwa
0p,,
l" ~
IFN-
VSV
IZ
O
O
FPV I
Z
O
~ so
Ix
0
U.I
--i
0
-J
0
.1
1
10
0
.1
1
10
100
IFN ADDED (IU/ml)
Fig. 2. Effect of natural IFN-a +/3 (Namalwa) and of natural IFN-y on plaque formation by fowl-plague virus and by VSV in human
embryonic foreskin cell cultures. IFN was given 18 h before virus challenge. Plaques were counted 48 h after infection. Plaque
formation by FPV and VSV were measured in parallel experiments using triplicate cultures for each dilution of 1FN. The data are the
average of two independent experiments.
Table 1
Sensitivity of influenza A viruses fowl plague (FPV) and WSN and of VSV to IFN action in cultures of human embryonic foreskin
cells
IFN
• IFN concentration in I U / m l (or in pg/ml)
for 50% reduction
Yield reduction procedure
Plaquing procedure
Natural
IFN-a +/3
(Namalwa)
IFN-y
Recombinant
IFN-aB
IFN-aD
IFN-aF
nd, not done.
VSV
FPV
1.6
6
1.3
5
0.7 (4.3)
45 (237)
2.3 (21)
0.2 (1.2)
10 (54)
0.9 (8.4)
VSV
WSN
1.4
nd
2
nd
0.2 (1.2)
nd
nd
0.6 (3.7)
nd
nd
210
similar sensitivity to each type of IFN. Similar
inhibition curves were obtained using the highly
purified recombinant IFNs -aB, -aD, and -aF (not
shown).
The concentrations of each IFN required for
obtaining 50% plaque reduction are presented in
Table 1. These values represent the average of at
least two independent experiments done with triplicate cultures. The level of sensitivity of influenza virus was consistently slightly higher than
that shown by VSV. The effectiveness of each IFN
was different on human cells, the lowest corresponding to recombinant I F N - a D which was in
accordance with results published previously [6,7].
The purpose of the next experiment was to
improve our understanding of the observed high
sensitivity of FPV to IFN action on an influenza
virus of human origin. The strain WSN of influenza A virus was found to replicate in H E F
cells although it was not a plaque former. The
yield of influenza virus replicated in H E F cells,
infected at an m.o.i, of 1 p.f.u./cell, was titered by
plaque formatin on calf-kidney primary cultures.
The results shown in the table confirmed the high
sensitivity of influenza virus to ! F N action in
human cells found by the plaquing procedure.
5. DISCUSSION
In this study it could be shown that influenza A
viruses are very sensitive to the action of natural
human IFN-a + fl and -T and to three subspecies
of recombinant IFN-a. The sensitivity of influenza
virus was comparable to that of VSV which was
taken as the reference. No specific antiviral activity was observed in any of the IFNs compared,
including the IFN-T. IFN-T operates in human
fibroblast cells by interaction with a class of cell
sul"face receptors distinct from that for IFNs a
and ft. Notwithstanding the similarity in the ef-
fects of IFN-a + fl and IFN-T on influenza virus
replication in human cells, it is quite possible that
each type of IFN operates by different mechanisms at the molecular level. This question is at
present under study in our laboratory. In contrast
to human embryonic fibroblast cells, the antiviral
action of mouse IFN-T against influenza virus and
VSV in mouse embryonic fibroblast cells is uncoupled [8,9]. In these cells IFN-T induces a potent
antiviral state against VSV but not against influenza virus. These results demonstrate that IFN-T
can induce different antiviral states depending on
the host species of the target cell.
ACKNOWLEDGEMENTS
We thank M. Gruetter for samples of pure
recombinant a interferons, W. Wiesendanger for
production of T interferon, and J.A.L. Auden for
his help in preparing the manuscript.
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