Bull Vet Inst Pulawy 55, 575-579, 2011
DIFFERENT PATTERNS OF MULTIPLICATION
OF EQUINE INFLUENZA VIRUS SEROTYPES H7N7
AND H3N8 IN CHICKEN EMBRYO
MAŁGORZATA KWAŚNIK, WOJCIECH ROŻEK, AND JAN F. ŻMUDZIŃSKI
Department of Virology,
National Veterinary Research Institute, 24-100 Pulawy, Poland
[email protected]
Received: October 28, 2011
Accepted: December 1, 2011
Abstract
The purpose of the study was to compare two models of multiplication patterns of equine influenza virus H7N7 – subtype
A1 and H3N8 – subtype A2, after inoculation into embryonated chicken eggs through the allantoic cavity and the yolk sack. The
relationship between the presence of haemagglutinin activity in allanto-amniotic fluid and the presence of M1 protein in
chorioallantoic membranes, vitteline membranes, and embryos body was determined. The distribution of equine influenza virus
multiplication sites in embryonated chicken eggs has revealed different patterns for A1 and A2 viruses. Virus subtype A1 multiplied
better after inoculation into the yolk sack than into the allantoic cavity. Virus subtype A2 multiplied equally well after inoculation
into the allantoic cavity as well as after inoculation into the yolk sack. There were cases with negative haemagglutination titers of
allanto-amniotic fluid of eggs inoculated into the yolk sack with both influenza virus subtypes, though the M1 protein was detected in
the vitteline membranes. Thus, the negative haemagglutination titres of allanto-amniotic fluid cannot be treated as the only indicator
of virus multiplication after inoculation into the yolk sack.
Key words: equine influenza virus, isolation, embryonated chicken eggs.
Equine influenza virus belongs to the
Orthomyxoviridae family type A and is represented by
two serotypes: H7N7 (subtype A1) and H3N8 (subtype
A2). The H7N7 strain has not been isolated from horses
since 1980. H3N8 strain circulates in equine population
throughout most of the world and it is regularly isolated
from horses in Europe (2, 3, 15, 21, 22). Similarly to
human influenza, equine influenza is a highly
contagious disease. Virus is spread via aerosolised
respiratory secretions. The risk of infection transmission
is increased by exchanging of horses between studs,
race-tracks, countries. The vaccines against equine
influenza infection do not provide the protection
comparable to protection stimulated after natural
infection (4, 21).
In order to isolate and multiply influenza virus
under laboratory conditions, embryonated chicken eggs
and cell cultures are used. The reverse transcription
polymerase chain reaction assay (RT-PCR) is the most
sensitive to detect the virus. Virus isolation in
embryonated chicken eggs is carried out routinely for
surveillance and vaccine strain selection and production
(5, 13, 21). The eggs are very efficient for multiplication
of the virus because of their sensitivity to infection and
metabolism intensity. Their advantages are: simple
method of inoculation, low costs of incubation, and
presence of allanto-amniotic fluid – easy and convenient
source of viral particles. However, there are difficulties
during isolation of wild-type strains of the virus in
embryonated chicken eggs. The cause of these problems
lay in a too small number of infective particles of the
virus in the material from infected animal, or late sample
collection. The purpose of the study was to compare two
models of virus inoculations: into the allantoic cavity
and into the yolk sack. The relationship between the
presence of haemagglutinin activity in the allantoamniotic fluid and the presence of M1 protein in
chorioallantoic membranes, vitteline membranes, and
embryos was also studied. Two subtypes of equine
influenza viruses (A1 and A2) were taken into
consideration in the analysis.
Material and Methods
Inoculation of embryonated chicken eggs.
Eighty 10-day-old SPF embryonated chicken eggs were
used for the experiment. Forty were infected with equine
influenza A1 virus and the others with A2 virus. Two
modes of inoculation were applied: into the allantoic
cavity and into the yolk sack. The infection dose 100
EID50 was used independently of the way of inoculation
and the subtype of influenza virus. The eggs were
incubated at 37°C up to 72 h after inoculation. Embryos,
576
which were dead after 48 h or 72 h of incubation, and
those that remained alive were cooled down to 4°C for
about 2 h. The allanto-amniotic fluids, the
chorioallantoic membranes, the vitteline membranes,
and embryo’s bodies were collected and stored at 20°C.
Haemagglutination assay (HA). HA was done
according to the standard of the World Organisation for
Animal Health (OIE) procedures described in the
Manual of Diagnostic Tests and Vaccines for Terrestrial
Animals (2009, vol. 2, section 2.5., chapter 2.5.7.)
Sample preparation for electrophoretic
analysis. Three volumes of sample buffer (60 mM TrisHCl, pH 6.8; 2% sodium dodecyl sulfate (SDS), 10%
glycerol, 0.001% bromphenol blue) was added to every
chorioallantoic membrane and vitteline membrane (3 ml
of buffer per 1g of membranes). The membranes were
disintegrated (Dounce homogeniser) at 4°C and heated
for 10 min at 95°C. Then the samples were sonicated (8
microns amplitude, 1 min) and centrifuged for 15 min
(4,500 rpm) at 4°C. The supernatants were saved for
electrophoretic analysis.
The embryo bodies were homogenised with one
volume of sample buffer (1 ml of buffer per 1g of
embryos). Then other 3 ml of sample buffer were added
to 1 ml of the sample. The next steps: heating,
sonication, and centrifugation were done as described
for membrane preparation.
The concentration of proteins in homogenates
of membranes and embryos was measured by
colorimetric assay (Dc Protein Assay, Bio-Rad). Thirty
micrograms of protein samples were used for
electrophoresis.
Electrophoresis
and
immunoblotting.
Proteins were separated with sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS PAGE) and
transferred to polyvinylidene fluoride (PVDF)
membrane. The membrane was blocked at room
temperature in buffer – 5% bovine serum albumin in
TBST (20 mM Tris-HCl, pH 7.4; 150 mM NaCl, 0.05%
1
22
Tween 20) for 12 h and next incubated with mouse
monoclonal antibody for M1 protein (Serotec) (1:
10,000) for 1 h at room temperature with constant
shaking. After incubation the membranes were washed
three times with TBST. Fragments (Fab’)2 of rabbit IgG,
specific for Fc fragments of mouse IgG labelled with
horseradish peroxidase (Jackson Immunoresearch) were
used. The conjugate was diluted 1:20,000 (in TBST) and
incubation of membrane was carried out for 30 min at
room temperature. After incubation, the membranes
were washed three times with TBST for 5 min at room
temperature. After reaction with a substrate (Amerham
ECL Plus Western Blotting Detection System) results of
the detection were visualised on X-ray film.
Results
After inoculation of influenza virus subtype A1
into allantoic cavity, M1 protein was detected in eight
out of 20 samples of chorioallantoic membranes (Fig.
1A). In the sample from vitteline membranes and the
embryo body, M1protein was not detected (Figs 1B,
1C). After inoculation of influenza virus subtype A2 into
allantoic cavity, M1 protein was detected in
chorioallantoic membranes in all samples of inoculated
chicken embryos (Fig. 1D), in three out of 20 samples of
vitteline membranes (Fig. 1E), and in five out of 20
samples of embryo bodies (Fig. 1F).
After inoculation of influenza virus subtype A1
into the yolk sack, M1 protein was detected in 10 out of
20 samples of chorioallantoic membranes and in 18 out
of 20 samples of vitteline membranes (Figs 2A, 2C).
The M1 protein was not detected in samples from
embryo bodies (Fig. 2B). After inoculation of influenza
virus subtype A2, M1 protein was detected in 18 out of
20 samples of chorioallantoic membranes, in 20 out of
20 samples of vitteline membranes, and not detected in
homogenates of embryo bodies (Figs 2D, 2E, 2F).
1
A
1
22
D
1
B
1
22
C
22
22
E
1
22
F
Fig. 1. Detection of M1 protein in particular parts of chicken embryo after inoculation into the allantoic cavity; by
immunoblotting technique. A, B, C - inoculation with equine influenza virus A1, and D, E, F - inoculation of equine
influenza virus A2; A, D – chorioallantoic membranes, B, E – embryos, C, F – vitteline membranes
1 - positive control (M1 protein), 2 – negative control (non infected chicken embryo), 3-22 test samples.
577
1
22
1
22
A
1
22
D
1
22
B
1
22
E
1
22
C
F
Fig. 2. Detection of M1 protein in particular parts of chicken embryo after inoculation into the yolk; by immunoblotting
technique. A, B, C - inoculation with equine influenza virus A1; D, E, F - inoculation of equine influenza virus A2; A,
D – chorioallantoic membranes; B, E – embryos; C, F – vitteline membranes; 1 - positive control (M1 protein); 2 –
negative control (non-infected chicken embryo); 3-22 test samples.
Table 1
Comparison of values of HA titers and M1 detection by immunoblottig technique
A1
A2
Inoculation into
Inoculation into yolk
Inoculation into
Inoculation into yolk
allantoic cavity
sack
allantoic cavity
sack
Geometric average
of HA titer of
2.4240
2.6925
2.0772
1.9734
allanto-amniotic
fluid
SD
0.2921
0.4316
0.1929
0.2122
P-value
<0.05
<0.1
M1
chorioallantoic
40%
50%
100%
90%
membranes
M1
25%
body of embryos
M1
vitteline membranes
90%
15%
100%
SD - standard deviation.
Results of HA of allanto-amniotic fluids of all
embryonated chicken eggs in reference to results of
detection of M1 proteins are presented in Table 1.
The highest HA titer demonstrated allantoamniotic fluids from chicken embryos infected with A1
subtype into the yolk sack. M1 protein was detected also
in 90% of vitteline membranes. In case of A2, the
highest HA titers were obtained after inoculation into
allantoic cavity and M1 protein was detected in 100% of
chorioallantoic membranes.
There were two cases of chicken embryos
inoculated into the yolk sack with A2 subtype when HA
titer of allanto-amniotic fluids were negative and the
chorioallantoic membranes did not reveal any viral
proteins, whereas M1 protein was detected in vitteline
membranes. The same result was obtained after
inoculation of A1 virus into the yolk sack.
Discussion
There are many factors influencing the isolation
of influenza virus in embryonated chicken eggs. The
first step essential for viral infection of cell is binding of
viral HA to host cell surface glycan receptors. Two main
factors: cell receptors specificity and viral HA structure
determine the effective viral infection (20). It has been
shown that different cell types contain different
amounts, types, and linkages of sugar chains by using
sialyl linkage-specific lectins (1, 7, 9, 11, 17). Cells
578
from particular parts of embryo show different
expression of N-glycans types. Recent studies, based on
the analysis with multi-dimentional high pressure liquid
chromatography (HPLC) mapping and matrix assisted
laser desorption ionisation time-of-flight mass
spectrometry (MALDI-TOF-MS), showed that molar
percentages of α 2-3 sialic acid (SA) linkage in
chorioallantoic and amniotic membranes were 27.2 and
15.4, respectively. Molar percentages of α 2-6 SA
linkage in chorioallantoic membranes were 8.3 and 14.2
in amniotic membranes (18). Thus, the affinity of HA to
cell receptors is characteristic and depends on the
particular species of animals. Human influenza viruses
prefer glycan receptors α 2-6 SA, so they are more
efficiently isolated by inoculating samples into the
amniotic, rather than the allantoic cavity of embryonated
chicken eggs (6, 10). Molar percentages of sulfated
glycans, recognised by human influenza virus, were 3.8
in chorioallantoic membranes and 12.7 in amniotic
membranes (18). Equine influenza virus similar to avian
influenza prefers α 2-3 SA, although the virus should
prefer chorioallantoic rather than amniotic membranes.
The study comprised inoculation of two strains of
equine influenza virus (A1 and A2) into the allantoic
cavity and into the yolk sack. The distribution of equine
influenza virus multiplication sites in embryonated
chicken eggs has revealed different patterns after
inoculation with A1 and A2 viruses. The data showed
that equine influenza virus subtype A1 multiplies
definitely better in embryonated chicken eggs after
inoculation into the yolk sack than into the allantoic
cavity. Equine influenza virus subtype A2 multiplies
equally well in embryonated chicken eggs after
inoculation into the allantoic cavity, and after
inoculation into the yolk sack. Inoculation of influenza
virus into the yolk sack seems to be the more effective
way for virus isolation than typical inoculation into the
allantoic cavity. Different ways of inoculation seems to
be the most effective method during isolation of viruses.
The trials of isolation of avian or swine influenza viruses
using three inoculation ways – into the allantoic cavity,
into the yolk sack, or onto the chorioallantoic membrane
showed that the inoculation into the yolk sack or onto
chorioallantoic membrane were the most effective.
Thus, combined use of three methods of virus
inoculation increases the number of virus isolations (8,
19, 23, 24).
The differences found during A1 and A2
subtype of equine influenza viruse multiplication may
come from the heterogeneity of HA structures. In case
of avian influenza viruses, the differences of virus
spread within chicken embryo are based on different
activation of the haemagglutinin by proteolitic cleavage.
The haemagglutinin of pathogenic strains is cleaved in
cells of each layer whereas the haemagglutinin of nonpathogenic strains is cleaved only in the allantoic
epithelium. Thus, the spread of non-pathogenic strains is
restricted in the embryonated chicken eggs (12, 14, 16).
The isolation of influenza virus in embryonated chicken
eggs is usually confirmed by haemagglutination assay of
allanto-amniotic fluid. The average haemagglutination
titers of allanto-amniotic fluids were higher in case of
inoculation with A1 subtype into the allantoic cavity as
well as into the yolk sack, in comparison to the
inoculation with A2 subtype. However, despite of the
presence of haemagglutinin in allanto-amniotic fluid
after infection with A1 subtype into allantoic cavity, M1
viral protein was detected only in 40% of chorioallantoic
membranes, and other elements of chicken embryo did
not show the presence of any viral proteins. The allantoamniotic fluids from chicken embryos infected with
influenza virus A2 subtype had lower haemagglutination
titer, than in case of A1 infection but viral proteins were
detected in 100% of chorioallantoic membranes and in
50% of embryo bodies.
M1 viral protein was not detected in most of the
analysed parts of embryo bodies after inoculation with
equine influenza virus subtype A1 into the allantoic
cavity in spite of the presence of haemagglutination
activity in the allanto-amniotic fluids.
In case of infection with influenza virus A2
subtype haemagglutination activity is considered as the
indicator of virus multiplication, and is confirmed by
M1 presence in the membranes of embryos. Two cases
with negative haemagglutination titres of allantoamniotic fluid of embryonated chicken eggs infected
into the yolk sack with influenza virus subtype A2 were
demonstrated. In chorioallantoic membranes there were
no viral proteins but in the vittaline membranes viral
proteins were detected. Detection of viral proteins in
vitteline membranes with negative haemagglutination
titers of allanto-amniotic fluid and lack of viral proteins
in the chorioallantoic membranes was also proved in
case of inoculation of A1 subtype. It follows the results
showing that negative haemagglutination titres of
allanto-amniotic fluid cannot be treated as the only
indicator of virus multiplication after inoculation into
the yolk sac. This observation is significant for the
interpretation of the results of strains isolation,
particularly when the results concern the specific
multiplication of some strains of influenza virus after
inoculation into the yolk sack.
Acknowledgments: The authors wish to
thank Mrs. Urszula Bocian for excellent technical
assistance. This work was supported by the Polish
Ministry of Science and Higher Education (2 P06K 006
29).
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