Conversion of MDCK cell line to suspension culture
by transfecting with human siat7e gene and its
application for influenza virus production
Chia Chua,b, Vladimir Lugovtsevc, Hana Goldingc, Michael Betenbaugha, and Joseph Shiloachb,1
aDepartment of Chemical and Biomolecular Engineering, Johns Hopkins University, 3400 N. Charles Street, Baltimore, MD 21218; andbBiotechnology Core
Laboratory, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, andcCenter for Biologics Evaluation and
Research, Food and Drug Administration, 9000 Rockville Pike, Bethesda, MD 20892
Communicated by John B. Robbins, National Institutes of Health, Bethesda, MD, July 14, 2009 (received for review March 10, 2009)
MDCK cells are currently being considered as an alternative to
embryonated eggs for influenza virus propagation and hemagglu-
tinin (HA) production intended for vaccine manufacturing. MDCK
to grow in suspension burdens the process of scale up and hence
their production capability. Anchorage-dependent MDCK cells
were converted to anchorage-independent cells, capable of grow-
ing in suspension as a result of transfection with the human siat7e
gene (ST6GalNac V). This gene was previously identified as having
an important role in cellular adhesion when the transcriptions of
genes from anchorage-dependent and anchorage-independent
HeLa cells were compared. Unlike the parental MDCK cells, the
siat7e-expressing cells were capable of growing in shake flasks as
suspension cultures, achieving maximum concentration of 7 ? 105
cells/mL while keeping close to 100% viability throughout the
growth phase. In production experiments, the siat7e-expressing
cells were infected with the Influenza B/Victoria/504/2000 strain. It
was determined that the cell-derived viruses retained similar
antigenic properties as those obtained from egg-derived viruses
and their nucleotide sequences were identical. The specific pro-
duction of hemagglutinin (expressed in hemagglutination units
times higher than the specific production from the parental MDCK
cells. If this suspension process scales up, the production potential
of HA from 10 L of siat7e-expressing cells at a concentration of 106
cells/mL would be equivalent to the amount of HA obtained from
10,000 embryonated eggs.
anchorage-independent ? hemagglutinin ? sialyltransferase ? vaccine
annually (1). In response to rapid antigenic drift in influenza
viruses, the most effective approach taken has been the distri-
bution of trivalent inactivated viral vaccines, which are tradi-
tionally produced in chicken embryonated eggs (2). However, in
the event of a pandemic outbreak, this egg-based production
system may not be adequate to meet the surge in demand quickly
enough. The limitations associated with egg-based vaccines,
which include reliable egg supplies, prolonged cultivation peri-
ods, and cumbersome operations have spurred exploration of
alternatives. Among the potential alternatives for vaccine pro-
duction, the use of characterized, immortalized cell lines (par-
ticularly MDCK, VERO, and PER.C6) has been investigated.
These cell lines have been found to produce consistently high
viral titers (3–8). Nevertheless, one of the limiting aspects in
scaling up the virus production in these continuous cell lines is
the fact that these cells are anchorage-dependent and thus
require surface adhesion to proliferate (9, 10). Without surface
attachment, these cells cannot exert their normal cyclin-
dependent kinase activity through the signaling cascades initial-
ized by interactions between integrins and extracellular matrix
(11–15). For industrial production in bioreactors, the required
nfluenza-related illnesses cause an estimated 100,000 hospi-
talizations and tens of thousands of deaths in the United States
surface area can be provided using microcarrier beads (16–19).
Although this approach is sufficient to obtain high virus pro-
duction yield (18, 19), this propagation strategy is cumbersome
compared with propagation of cells in suspension. An MDCK
the scale-up process of influenza virus production.
In a previous study we compared the transcription profiles of
anchorage-dependent and anchorage-independent HeLa cells
using DNA microarrays (20). The gene siat7e (ST6GalNac V)
was identified as one of the genes that play a role in controlling
the degree of cell adhesion. It was shown that higher siat7e
transcription corresponded to a lower degree of adhesion by
microscopic evaluation and by monitoring cell detachment in a
siRNA was followed by enhanced adhesion. The human sialyl-
transferase ST6GalNac V, a member of the ST6GalNac family
of sialyltransferases, is a type II Golgi membrane protein that
transfers sialic acid from the donor CMP-Neu5Ac to the GalNac
residue on the ganglioside, GM1b, forming GD1?. Tsuchida et
al. (21) proposed indirect involvement of siat7e in synthesizing
disialyl Lea, a carbohydrate structure conjugated to proteins and
ceramides on the cell surface. In other studies, glycosphingolip-
ids including gangliosides have been reported to mediate cell
adhesion through the sugar residue interactions in the glycosyn-
apse microdomains (22, 23). These reports are consistent with
our findings on the relationship between siat7e gene expression
producers of several viruses including influenza A and B viruses.
The conversion of these anchorage-dependent cells to cells
capable of growing in suspension will simplify the production
process and has the potential to supplant current production
procedures in chicken embryonated eggs. In the present work we
report on the transfection of the anchorage-dependent MDCK
cells with the human siat7e gene, on the properties of the
siat7e-expressing cells and on their capability to produce the
Transfection of MDCK Cells with Human siat7e and Its Effects on
Cell–Cell Adhesion and Cell Spreading. Anchorage-dependent
MDCK cells exhibited changes in cell-cell adhesion and cell
spreading behavior following the incorporation of the human
siat7e gene as shown in Fig. 1. Cells transfected with the siat7e
shown in Fig. 1B (clone 1) and C (clone 2) appear to spread less
on the cell culture flask than the parental cells shown in Fig. 1A;
the siat7e-expressing cells also lost their ability to form a tight
Author contributions: C.C., M.B., and J.S. designed research; C.C. and V.L. performed
research; C.C., V.L., H.G., M.B., and J.S. analyzed data; and C.C., V.L., and J.S. wrote the
The authors declare no conflict of interest.
Freely available online through the PNAS open access option.
1To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
September 1, 2009 ?
vol. 106 ?
junctions with the neighboring cells. It was also observed that
when the siat7e-expressing cells undergo prolonged culture,
some cells would self-detach while maintaining their viability.
Assessment of transfection efficiencies with the siat7e plasmid
using the FACSCalibur machine showed that approximately 4%
of MDCK cells were transfected 24 h after introducing the
Gene Expression Differences Between the Parental and the siat7e-
Expressing MDCK Cells. The detection of the human siat7e mRNA
in the parental and the siat7e-expressing cells and the expression
of a housekeeping gene (endogenous GAPDH) are seen in Fig.
2A. It is clear that there is expression of human siat7e in the
transfected cells but no expression in the parental cells, while
GADPH expression was detected in all samples. Real-time PCR
was performed to quantify the expression of siat7e and the
expression of the housekeeping gene in clones 1 and 2 (Fig. 2B).
The increase in the siat7e expression was correlated with the
degree of cell-cell adhesion and cell spreading of these two
transfected clones seen in Fig. 1.
Surface Charge Differences Between the Parental and the siat7e-
Expressing MDCK Cells. To assess cell surface differences between
the two cell lines, the cell surface charge was measured using
FITC-labeled cationized ferritin (24–26). Interactions between
cationized ferritin and negative charge sites on the cell surface
the amount of bound ferritin molecules (25). The signal profiles
from each cell line, with and without ferritin treatment, are
shown in Fig. 3. Flow cytometric analysis showed a shift in the
overall signal distribution of the siat7e-expressing cells (Fig. 3B).
The shift indicates higher signal intensities emitted from the
fluorescein (FITC), which should correspond to higher number
of anionic sites on the membrane surface. No difference was
observed when the ferritin was not present (Fig. 3A).
Growth Kinetics in Monolayer and Suspension of the Parental and
siat7e-Expressing MDCK Cells. Growth, viability, glucose consump-
tion and lactate production of the parental and the siat7e-
expressing MDCK cells grown as a monolayer in T flasks are
shown in Fig. 4 A–C and as suspension culture in Fig. 4 D–F. The
siat7e-expressing cells grew less than the parental cells in the T
flask (Fig. 4A). Their density reached 7 ? 104cells/cm2com-
pared to 2 ? 105cells/cm2of the parental cells after 179 h of
growth, although the percent viability of the cells was similar
(Fig. 4B). Glucose consumption and lactate production in the
two cell lines were similar until the siat7e-expressing cells
approached peak density in the T flasks as shown in Fig. 4C.
Parental MDCK cells. (B) Clone 1, isolated from the siat7e-expressing pool. (C)
Clone 2, isolated from the siat7e-expressing pool.
Parental and siat7e-expressing MDCK cells grown in T flasks. (A)
parental MDCK and in clones 1 and 2 of the siat7e-expressing cells. (A)
End-point RT-PCR. (B) Real-time PCR.
mRNA expression of human siat7e and endogenous GAPDH in
siat7e-expressing MDCK cells with and without ferritin. (A) Without ferritin
treatment; (B) with ferritin treatment. Parental MDCK cells (gray line), siat7e-
expressing cells (black line).
FITC signal distribution obtained by FACS analysis of parental and
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September 1, 2009 ?
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no. 35 ?
Opposite growth trends were observed when the two types of
cells were propagated in shake flasks. The growth curve (Fig.
4D) demonstrates that siat7e-expressing cells were able to pro-
liferate in suspension culture, whereas the parental cells could
not. The siat7e-expressing cells grew exponentially to a concen-
tration of 7 ? 105cells/mL. High viabilities (Fig. 4E) of the
siat7e-expressing cells were seen throughout the 12-day growth.
These cells were at least 90% viable while the viability of the
parental MDCK cells declined steadily over the culture period.
The glucose and lactate profiles shown in Fig. 4F indicate that
parental MDCK cells consumed more glucose and produced
more lactate than the siat7e-expressing cells especially at later
culture times when cell densities were greater in the siat7e-
expressing cells. Microscopic analysis at the end of the growth
showed that the surviving parental MDCK cells were aggregated
in large clumps, while the siat7e-expressing cells, on the other
hand, appeared healthy and were suspended primarily as indi-
Influenza Virus Growth and HA Titer in Parental and siat7e-Expressing
MDCK Cells. The yield of influenza virus in parental and siat7e-
expressing MDCK cells was evaluated by analysis of growth
kinetics of a model virus B/Victoria/504/2000 per 106cells.
Summarized in Table 1 are the highest values of both the viral
and the HA titers. The values were obtained 36 to 48 h post
infection in the case of the adherent cells and 24–38 h in the case
of cells grown in suspension. The viral infectivity titers, ex-
pressed as 50% egg infectious dose per mL (EID50/mL), were
similar in three growth conditions: monolayer culture of the
anchorage-dependent parental MDCK cells, monolayer culture
of the siat7e-expressing cells and the siat7e-expressing cells
grown in suspension. However, remarkable differences were
observed for HA titers, expressed in hemagglutinating units
(HAU). When calculated per 106cells, 2,155 HAU was obtained
from the parental MDCK cells, 8,606 HAU from the siat7e-
expressing cells grown in monolayer, and 54,348 HAU from the
siat7e-expressing cells grown in suspension in shake flasks.
Shown in Fig. 5 is the cell viability of the infected siat7e-
expressing cells grown in suspension and the HA titers over the
time course of one representative kinetic experiment.
Virus Antigenic Stability During Replication in Parental MDCK Cells
and siat7e-Expressing Cells. The effect of different cell substrates
on virus antigenic properties was evaluated in hemagglutination
inhibition test (HAI). The HAI titers of three ferret sera that
were infected with egg-grown reference virus B/Victoria/504/
in viable cell density (VCD). (E) Viability %. (F) Glucose consumption and lactate production (shaded) in g/L.
Table 1. Virus titers in different cell substrates
Viral titer Virus titer per 106cells
HAU EID50, log10
siat7e-expressing cells monolayer
siat7e-expressing cells* suspension
8.35 ? 0.17
6.90 ? 0.12
7.87 ? 0.12
Influenza strain B/Victoria/504/2000 was used to infect the substrates between M.O.I.s of 1.0 and 2.0 TCID50.
Hemagglutinin titers and infectious titers were measured using supernatant from whole cell lysate samples.
*Cells were infected at 107/mL density in suspension culture and then diluted to 106/mL for propagation.
www.pnas.org?cgi?doi?10.1073?pnas.0905912106Chu et al.
parental MDCK cells and the siat7e-expressing MDCK cells
grown either in monolayers or in suspension. The results are
shown in Table 2. In all cases, the sera titers were within two-fold
difference, demonstrating that cell-derived viruses were as an-
tigenic as those obtained from the egg-derived reference virus.
Direct DNA sequencing of RT-PCR products amplified from
HA and NA (neuraminidase) viral gene segments, showed that
the cell-derived viruses and the egg-derived reference virus had
identical nucleotide sequences. These data demonstrate that
replication of the virus in parental or siat7e-expressing cells did
not alter the antigenic properties of the virus.
In a previous study, we identified two genes that have a role in cell
adhesion (20): siat7e, a type II membrane glycosylating sialyltrans-
ferase, and lama4 which encodes laminin ?4, a member of the
laminin family of glycoproteins. These two genes were identified
following a comparison of gene transcription of two phenotypically
distinct HeLa cells, anchorage-dependent and anchorage-
independent. It was demonstrated that decreased expression of
siat7e in the anchorage-independent HeLa cells, or enhanced
expression of the lama4, resulted in greater aggregation and
morphological changes compared with the untreated anchorage-
independent HeLa cells. An opposite effect was observed when
decreased in the anchorage-dependent HeLa cells.
Engineering cell lines to improve biotechnology processes was
one of our major aims of the previous study. One of the important
commercial production processes that are still in need of a well-
is currently being produced in embryonated eggs (27). Since the
production in eggs is quite cumbersome and time consuming,
replacing the embryonated eggs process with mammalian cells,
especially MDCK cells, is an appealing option (4, 6, 8). Since
MDCK cells are anchorage-dependent, the replacement of the
embryonated eggs with these cells will introduce additional pro-
cessing difficulty. Specifically, these processes often require inclu-
sion of microcarriers, which are difficult to scale-up. Microcarriers
require multiple additional processing steps including seeding of
cells on a surface for subsequent growth (which may be limited by
surface area), maintenance of the microcarriers in the bioreactor
Not surprising, current production of monoclonal antibodies and
most other biopharmaceuticals by mammalian culture utilizes cell
lines such as Chinese Hamster Ovary (CHO), Baby Hamster
of the MDCK cells to grow in suspension would considerably
simplify the production process of influenza vaccine. Although the
current state-of-art has already used suspension MDCK cell cul-
tures to produce influenza vaccines (28), we report the targeted
engineering of the MDCK cell lines to anchorage-independent
culture while others relied on long serial passaging approach.
Incorporation of the human siat7e gene into the MDCK cells,
as demonstrated in this study resulted in their conversion to
anchorage-independent cells. The cells grew well in suspension
in shake flasks. The cultures reached a concentration of 7 ? 105
cells/mL maintaining at least 90% viability throughout the
growth period. The human gene was successfully incorporated
and transcribed, (Fig. 2A) modifying considerably the cell
phenotype. By using CF-FITC it was possible to determine that
there is a change in the net charge on the surface of the
siat7e-expressing cells. The increased negative charge may be
associated with the increased number of sialic acids moieties
attached to the cell surface gangliosides by siat7e. Elevated
negative charge of the cell surface may contribute to a decreased
cell-to-surface adhesion and to electrostatic repulsion between
cells, and thus allowing the cells to grow in suspension.
Siat7e-expressing cells were not only able to grow in suspen-
sion and to produce identical virus as the one produced in
embryonated eggs, their specific production of HA was about 20
times higher than the anchorage-dependent parental cells. Re-
cent publications have reported on the expression of the human
siat1 gene (ST6Gal I) in MDCK cells that was associated with an
increase in ?2,6-linked sialic acid on glycoproteins (29–31). The
researchers wanted to increase the number of 6-linked sialic
acids on the cell surface to enhance influenza virus sensitivity to
neuraminidase inhibitor, which is the key component of antiviral
drugs for influenza. In addition, a two-log increase in the
production of human influenza viruses in these cells was re-
ported (29). Unlike ST6Gal I, ST6GalNac V expressed in this
current study is responsible for adding sialic acid to the GalNac
residue located on the side position of oligosaccharide chains
instead of the common terminal position on the Gal residue. The
infectivity with the B/Victoria/504/2000 of the siat7e-expressing
cells measured as EID50/cell was found to be slightly lower than
that of the parental cells but the HA production was found to be
higher. In a seasonal influenza vaccine, a typical dose is com-
posed of 15 ?g purified HA from each of the three selected
influenza strains (H1N1, H3N2, and B). Since the vaccine is
of these cells. In work conducted by Tree et al., production of
influenza virus A strain in MDCK cells grown on various types
of microcarriers were compared to chicken eggs. They reported
and 2.0 ? 105HAU/mL in chicken eggs. Based on this infor-
mation, they estimated that 1,000 L MDCK cells grown on solid
microcarriers would be equivalent to roughly 30,000 eggs, or 1 L
of siat7e-expressing MDCK cells without infection are also shown (open
Table 2. HAI titers with viruses from different cell substrates
Sera 1Sera 2 Sera 3
siat7e-expressing cells monolayer
Sera were obtained from three ferrets 3 weeks after intranasal infection
with egg-derived reference virus B/Victoria/504/2000.
*Reciprocal of the highest dilution of serum capable of completely inhibiting
HA activity of the respective virus. Data are from a single representative
†Cells were infected at 107/mL density in suspension culture and then diluted
to 106/mL for propagation.
Chu et al.PNAS ?
September 1, 2009 ?
vol. 106 ?
no. 35 ?
would be equivalent to about 30 eggs. In another study, titer of
2.5 ? 103HAU/mL was obtained in a stirred tank reactor using
Cytodex I microcarriers (16) and a maximum titer of approxi-
mately 4.0 ? 104HAU/mL was obtained in WAVE cellbags
using Cytodex I microcarriers (18). It is important to note that
in these studies the Influenza A virus strain was used. For the
Influenza B strains, which was used in our study, the HA titers
are commonly on the scale of 100 (32, 33). Based on the HA
production capability, we estimate that 1 mL of culture contain-
ing 106cells can produce approximately 40,000 HAU. Taking
into consideration that the average production of HAU per egg
is also around 40,000, 10-L culture of the siat7e-expressing
MDCK cells can produce the equivalent amount of HAU
produced in 10,000 embryonated eggs. These data demonstrate
that the established siat7e-expressing MDCK cell line has the
potential to significantly increase the efficiency of manufacture
of influenza vaccines, and thus, quite possibly contributing to
lower vaccine cost and wider availability to a greater number of
recipients worldwide. In the immediate future, it is imperative
that we take on a systems approach, as demonstrated elsewhere
(34), to integrate strain improvement, upstream optimization,
and downstream processing to further improve our production
strategy. A fully developed and well-characterized cell substrate
system would be advantageous not only economically but also
presents a stronger case for approval by federal regulation
Materials and Methods
Cell Line and Virus. Madin Darby Canine Kidney (MDCK) cells were acquired
grown in 37 °C, 5% CO2humid incubator using Minimal Essential Medium
containing Earl’s salts and L-glutamine (Invitrogen) and supplemented with
Fetal Bovine Serum (Invitrogen) to a final concentration of 10%. Only cells
B/Victoria/504/2000 was obtained from the influenza virus depository of the
Center of Biologics Evaluations and Research, Food and Drugs Administration
competent cells (Invitrogen) were transformed with full-length human siat7e
gene expression vector (Cat. No. EX-V1581-M03, Genecopoeia). The plasmids
were purified using the QIAprep Spin Miniprep kit (Qiagen) and were used to
cells were seeded at 2 ? 105cells/well in a 24-well plate; day 2: 0.8 ?g plasmid
DNA was mixed with 2.0 ?L Lipofectamine 2000 and incubated together with
and suspended in growth medium; day 3: G418 was added to the growth
medium at a final concentration of 0.400 mg/mL, and the medium containing
G418 (selective medium) was routinely replaced every 3 to 4 days for a period
of 3 weeks. Stably transfected pool of siat7e-expressing cells were grown and
banked. Finally, clones were isolated by limiting dilution in a 96-well plate.
Gene Expression. RNA samples were isolated from parental MDCK cells and
from clones of the siat7e-expressing cells using RNeasy Total RNA Isolation kit
(Qiagen). SuperScript One-Step RT-PCR kit (Invitrogen) was used for the
reverse transcription and for PCR amplification experiments in accordance to
the manufacturer’s protocol, using the sense primer sequence 5?-ttactcgcca-
caagatgctg-3? and antisense primer sequence 5?-gcaccatgccataaacattg-3?.
sense primer sequence 5?-aacatcatccctgcttccac-3? and antisense primer se-
quence 5?-gaccacctggtcctcagtgt-3?. Briefly: cDNA synthesis was performed at
50 °C for 30 min, samples were incubated at 94 °C for 2 min to ‘‘hot-start’’ the
94 °C for 15 s annealing at 55 °C for 30 s, and extending at 72 °C for 10 s (14
s for the endogenous control). The target genes were amplified for 35 cycles
with a final extension at 72 °C for 10 min. The end products were resolved on
Kit (Applied Biosystems) with the same primer sequences described above.
Briefly: cDNA samples were synthesized from 0.5 ng RNA sample and ampli-
fied under standard thermal cycler protocol (50 °C for 2 min, 95 °C for 10 min,
and 40 cycles of 95 °C for 15 s and 60 °C for 1 min). Target Ct values were
averaged from replicates and fold changes were calculated against the en-
dogenous control, GAPDH.
Cationized Ferritin Binding Assay. Cationized ferrtin (Electron Microscopy
Sciences) was conjugated with FITC using the FITC Protein Labeling kit (Pierce
Biotechnology). Briefly: cationized ferritin was dialyzed with the supplied
borate buffer and incubated with FITC solution at room temperature for 1 h.
Excess FITC dye was removed using a dialysis cassette (Pierce Biotechnology).
Conjugated ferritin complex was quantified using E270 nm1%? 79.9 and
MW ? 750,000 for native ferritin and a correction factor of 0.3 for FITC whose
1 ? 107cells were detached from culture flasks using Hank’s-based cell
dissociation buffer (Invitrogen) and washed with PBS before re-suspending in
1 mL PBS containing FITC-conjugated ferritin at 50 ?g/mL final concentration
(24–26). The mixture was incubated on a thermomixer at 4 °C for 1 h and
The cells were immediately analyzed using the FACSCalibur flow cytometer.
Growth Kinetics. For growth kinetics in anchorage-dependent manner, pa-
105cells per one 25-cm2culture flask; 21 flasks were seeded for each cell line.
Glucose and lactate concentrations were measured using the YSI 2700 Select
biochemistry analyzer (YSI Life Sciences) and cell count was measured using
Cedex (Innovatis AG). Measurements were taken daily from three flasks. For
growth kinetics in suspension culture, cells from each line were seeded at
approximately 2 ? 105cell/mL in three 125-mL vented shake flasks containing
and shaken at 90 RPM. Measurements were taken at 48 h intervals.
Virus Growth Evaluations in Monolayer and Suspension Culture. Monolayer
culture: Parental MDCK cells or siat7e-expressing cells were grown to conflu-
ency in 25-cm2flasks (Corning). After removal of the growth media, the cells
were washed once with serum-free medium and the virus was added to each
flask at a multiplicity of infection (MOI) of 2.0 TCID50(50% tissue culture
infectious dose). After adsorption for 1 h at 37 °C, the cells were washed with
added. The infected cells were incubated at 33 °C for the remainder of the
experiment. Cell condition (appearance of cytopathogenic effect) was con-
stantly monitored and samples were collected every 8 h for virus infectivity
and hemagglutination (HA) titers determination.
Suspension culture: siat7e-expressing cells grown in shake flasks were
concentrated by centrifugation (600 rcf for 5 min) and re-suspended in a
serum-free medium at a density of 107cells/mL. After infection with the
influenza virus at an MOI of 2.0 TCID50, the cell suspension was incubated at
constant shaking at 37 °C for 1 h. At this time, the cells were precipitated and
suspended in DMEM supplemented with 10% FBS to a density of 106cells/mL.
The infected cells were incubated at 33 °C in the same conditions for the
remainder of the experiment; the controlled culture was treated in the same
way but without addition of the virus. Samples were taken every 8 h during a
period of 4 days and stored in aliquots at ?70 °C for virus infectivity titer and
HA titer determination. Cell concentration, viability and metabolic parame-
ters were monitored at each time point.
Determination of Virus Yield. Virus growth and concentration were deter-
mined by infectivity titer in chicken embryonated eggs (EID50) and by HA titer
using standard techniques described earlier (35–37).
Determination of Virus Stability During Replication in MDCK Cells. Antigenic
properties of the progeny virus harvested from the parental or the siat7e-
expressing cells (56 h post infection) were characterized by hemagglutination
inhibition test (HAI test) using a set of three homologous ferret antisera specific
to strain B/Victoria/504/2000. The HAI test was performed in 96-well plates (two
replicates for each serum sample) using 0.5% chicken red blood cells in PBS (pH
7.2) (37). Two viruses were considered antigenically indistinguishable if the
corresponding HAI titers did not exceed two-fold difference. In addition the
nucleotide sequences of viral gene segments encoding viral surface glycopro-
teins, HA and NA, were determined by direct DNA-sequencing of the RT-PCR
products and compared with those of the parental virus stock.
ACKNOWLEDGMENTS. We thank Dr. Bruce Raaka for his assistance with the
aspects of the experiments and formulation of the manuscript. This work was
Digestive and Kidney Diseases, National Institutes of Health.
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