Infectious bursal disease virus is an icosahedral
polyploid dsRNA virus
Daniel Luquea, Germa ´n Rivasb, Carlos Alfonsob, Jose ´ L. Carrascosaa, Jose ´ F. Rodríguezc, and Jose ´ R. Casto ´na,1
Departments ofaStructure of Macromolecules andcMolecular and Cellular Biology, Centro Nacional de Biotecnología/CSIC, Cantoblanco, 28049 Madrid,
Spain; andbCentro de Investigaciones Biolo ´gicas/CSIC, 28006 Madrid, Spain
Edited by Peter Palese, Mount Sinai School of Medicine, New York, NY, and approved December 11, 2008 (received for review August 27, 2008)
Viruses are a paradigm of the economy of genome resources,
reflected in their multiplication strategy and for their own struc-
ture. Although there is enormous structural diversity, the viral
genome is always enclosed within a proteinaceous coat, and most
virus species are haploid; the only exception to this rule are the
highly pleomorphic enveloped viruses. We performed an in-depth
characterization of infectious bursal disease virus (IBDV), a non-
enveloped icosahedral dsRNA virus with a bisegmented genome.
Up to 6 natural populations can be purified, which share a similar
particle density increases. Stoichiometry analysis of their genome
number of dsRNA segments inside the viral capsid. This is a
demonstration of a functional polyploid icosahedral dsRNA virus.
We show that IBDV particles with greater genome copy number
have higher infectivity rates. Our results show an unprecedented
replicative strategy for dsRNA viruses and suggest that birnavi-
ruses are living viral entities encompassing numerous functional
and structural characteristics of positive and negative ssRNA viruses.
cargo volume ? IBDV ? icosahedral dsRNA viruses ? life cycle ?
(ss) RNA, ssDNA, double-stranded (ds) RNA or dsDNA]. An
important limitation to the size of the viral genome is its
container, the protein capsid. Virus particles have evolved to
incorporate only a single copy of their genome within the protein
shell, a widespread but not universal strategy.
Capsid arrangement of 1 or more protein subunits defines the
volume available for the viral genome (1). Helical viruses are
considered open structures, since a genome of any size can be
enclosed by varying helix length. Icosahedral viral shells form a
container of limited internal volume for nucleic acid packaging.
Retroviruses represent a structural variation with a fullerene-
based capsid and are exceptional, since each virion particle
incorporates 2 copies of the positive-sense ssRNA (2). Envel-
oped pleomorphic viruses with helical nucleocapsids (encom-
passing negative ssRNA) have relaxed constraints on packaged
genome size (3), leading to the incorporation of additional RNA
segments, as in the case of influenza virus, an orthomyxovirus
with a segmented genome (4). Furthermore, paramyxoviruses
(with non-segmented genomes) are extremely pleomorphic and
can package several genomes efficiently; paramyxoviruses might
therefore be considered polyploid (5).
Most dsDNA, ssRNA, and dsRNA viruses are spherical and
build a defined cargo space, with sizes ranging from 20 nm to
of compaction, compatible with the inner particle dimensions
(7). Although variable nucleic acid densities are reported, it is
clear that there are upper (and lower) limits for the size of the
nucleic acid to be encapsidated.
Here, we analyzed the biophysical and biochemical properties
of a non-enveloped icosahedral dsRNA virus, the infectious
bursal disease virus (IBDV), an avian virus of the family
iruses are entities with limited coding capacity, but with
extensive diversity in their genetic material [single-stranded
Birnaviridae (8). IBDV has a bipartite dsRNA genome (seg-
ments A and B) that is packaged into a single virus particle, ?70
nm in diameter, exhibiting T ? 13 levo symmetry (9, 10).
Considering that the IBDV capsid average internal radius is 26.5
nm (9, 11), which implies a cargo space of ?77,900 nm3, and that
the dsRNA average size is 6 kpb, a genomic packing density of
?10 bp/100 nm3is obtained. This value contrasts with those
previously determined for icosahedral viruses with densities
?40 bp/100 nm3.
IBDV Segment A (3.2 kpb) has 2 partially overlapping open
reading frames (ORFs). The smaller of the two encodes VP5, a
nonstructural host membrane-associated protein; the larger
encodes a polyprotein that is cotranslationally processed by the
viral protease VP4 (12), yielding the capsid precursor protein
pVP2, as well as VP3 and VP4. pVP2 is further processed at its
C-terminal region to give the mature VP2. Segment B (2.8 kpb)
is monocistronic and encodes VP1, the RNA-dependent RNA
polymerase [RdRp (13)], which is packaged inside the virion.
VP2 and a variable amount of pVP2 assemble into 260 trimers
to form the capsid structural units (accounting for ?38 MDa);
VP3 is a multifunctional protein that interacts with itself (14),
pVP2 (11), VP1 (15, 16), and with the dsRNA to make ribonu-
cleoprotein complexes (17), a unique feature among dsRNA
IBDV is an icosahedral virus that can package more than 1
complete genome copy. Moreover, multiploid IBDV particles
propagate with higher efficiency than haploid virions. These
features suggest that IBDV and, indeed, all birnaviruses have a
number of structural and functional features (18, 19) that
represent evolutionary links to unrelated viruses.
Purification of IBDV Natural Populations. Despite the considerable
detail available about the high resolution structure of most
IBDV proteins, biochemical quantitation of its components is
limited to sodium dodecyl sulfate/polyacrylamide gel electro-
phoresis (SDS/PAGE) analysis and Coomassie blue staining;
proportion to their molecular weight (20). Several bands are
visible when IBDV virions, isolated from infected cultured cells
or from the bursa of Fabricius from infected birds, are purified
in CsCl gradients (21, 22). To determine the number of copies
of IBDV structural proteins with accuracy, we purified IBDV
virions by ultracentrifugation in CsCl gradients. At least 6 major
bands were visible and termed E1 to E6 from top to bottom (Fig.
Author contributions: D.L., G.R., C.A., J.L.C., J.F.R., and J.R.C. designed the research; D.L.,
G.R., C.A., J.F.R., and J.R.C. performed the research; D.L., G.R., C.A., J.L.C., J.F.R., and J.R.C.
analyzed the data; and J.R.C. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2009 by The National Academy of Sciences of the USA
February 17, 2009 ?
vol. 106 ?
cells were inoculated using E3–E6 fractions individually, the
same 6 major populations were recapitulated (data not shown).
These fractions were analyzed by SDS/PAGE (Fig. 1B) and
gel, it could be deduced that whereas E2–E6 fractions had
similar protein profiles for structural proteins VP1, pVP2 and
of pVP2 and contained no visible VP1. Electron microscopy
analysis of the E2–E6 fractions showed homogeneous popula-
tions of 65- to 70-nm diameter icosahedral particles that corre-
spond to T ? 13 capsids (Fig. 1C, E3 and E5). In the E1 fraction,
however, we observed complete virion-like particles, partially
broken particles, and isometric assemblies (11) whose size
corresponded to T ? 1 (?23 nm) and T ? 7 (?53 nm) particles
(Fig. 1C, E1). The colocalization of these structures indicated
that they have the same protein:RNA ratio. Moreover, consid-
ering that T ? 1 capsids are built only of VP2 trimers (10), E1
fraction isometric assemblies must be devoid of nucleic acids.
Due to its structural heterogeneity, the E1 fraction was excluded
from further analyses.
Stoichiometry of IBDV Structural Components. To determine
whether E2–E6 capsids are biochemically identical, we used
autoradiograms of SDS/PAGE gels to quantitate the structural
proteins in [35S]Met-labeled preparations of all 5 types of
particles (Table 1; supporting information (SI) Fig. S1).
E3–E6, supporting a similar protein composition for these
populations independently of their buoyant density. Our calcu-
lations identified 12 copies of VP1/virion, an unexpectedly large
copy number for the RdRp if we considerer that the IBDV
of the family Reoviridae, whose genome consists of 10–12
segments of dsRNA packaged within the same particle, thus
at a relatively constant amount, although it is not icosahedrally
ordered. Finally, the viral protease VP4 can be considered as
another structural component, although it is found in highly
For comparative purposes, we also performed stoichiometric
analysis based on Coomassie blue-stained gels, as described in
ref. 20. We obtained a systematic overestimation of copy ratios
for proteins VP1 and VP3, indicating preferential staining of
these polypeptides (Fig. S1).
We also analyzed the nucleic acid component of the distinct
IBDV populations from [33P]HPO4
ons to determine whether the differences in buoyant density
capsid. E3–E6 IBDV populations, which are infectious (see
below) and have a similar protein composition, and the E2
population were analyzed by agarose gel electrophoresis and
autoradiographed at the same protein concentration using intact
(Fig. 2A) or SDS- and proteinase K-treated virions (Fig. 2B).
The existence of a single band of defined mobility demonstrated
the structural integrity of the virion particles E2–E6 (Fig. 2A,
labeled as vRNA). Purified dsRNA segments A and B, obtained
after SDS and proteinase K treatments, were observed as 2
well-defined bands (Fig. 2B, labeled as dsRNA-A and dsRNA-
B). At equal particle concentrations, the amount of viral
genomic dsRNA incorporated into the virion capsid increased
from lower-density (E2) to higher-density (E6) IBDV popula-
tions, with equimolar amounts of segments A and B (Fig. 2B).
These differences in radioactive content corresponded to dif-
ferent amounts of packaged genomic segments, since interme-
diate size segments were not detected; consequently, we nor-
malized incorporated radioactivity relative to that of the E2
fraction (Fig. 2C). The data showed that the respective genome
content of E3, E4, E5, and E6 populations are, respectively, 2-,
3-, 4-, and 4-fold higher than that of the E2 population. In this
model, the number of packaged dsRNA segments is a natural
integer of those ratios, and the IBDV particle is therefore able
to package at least 4 dsRNA segments.
?2-metabolically labeled viri-
Biophysical Analysis of IBDV Populations. To obtain an independent
estimate of the molecular weight of the E2–E6 populations, and
to help interpret the quantitative biochemical results, we used
gradient for IBDV purification illuminated from the bottom after centrifuga-
tion to equilibrium, containing at least 6 IBDV fractions (denoted E1 to E6,
from top to bottom). E1–E6 bands represent 3, 5, 12, 16, 48, and 16% of the
total virion particles. (B) E1–E6 bands were collected by side puncture, ana-
lyzed by SDS/PAGE and developed by Coomassie staining. IBDV structural
proteins are indicated. (C) Electron microscopy of purified E1–E6 populations
negatively stained with 2% uranyl acetate. T ? 7 capsid-like (arrows) and T ?
bar, 100 nm.)
Purification of IBDV natural populations. (A) A typical CsCl linear
Table 1. Stoichiometry of IBDV polypeptides and dsRNA based on35S labeling
(Met)E2 E3 E4E5E6
7 ? 0.7
191 ? 13
589 ? 79
415 ? 30
27 ? 2
9 ? 1.4
75 ? 13
705 ? 54
487 ? 42
33 ? 4
13 ? 0.8
63 ? 5
717 ? 59
470 ? 55
60 ? 3
16 ? 1
37 ? 3
743 ? 23
468 ? 24
61 ? 11
15 ? 1.6
43 ? 3
737 ? 42
401 ? 50
24 ? 3
13 ? 3
55 ? 17
725 ? 41
457 ? 50
45 ? 18
41 ? 6
96 ? 12
684 ? 28
601 ? 60
50 ? 20
*These values indicate the corresponding normalized average copy number of IBDV structural proteins/capsid for each population
†Average copy number estimated from Coomassie blue-stained gels. These values are similar to previous analysis (20).
Luque et al.PNAS ?
February 17, 2009 ?
vol. 106 ?
no. 7 ?
analytical ultracentrifugation to study equivalent samples of the
IBDV populations (Fig. S2). Most E2–E6 IBDV particles sedi-
mented as a defined species with standard sedimentation coef-
ficient values (s20,w) of 290, 336, 376, 418, and 416 S, respectively
(Table 2). Dynamic light scattering experiments with the same
viral particles gave similar standard translational diffusion co-
efficients D20,w (Table 2), compatible with a main scattering
species for each IBDV population. From the combined s20,wand
D20,wcoefficients, the Svedberg equation allows determination
of the molar mass of the E2–E6 viral particles in a shape-
independent manner (23). We established that the main species
of E2–E6 particles have molar masses of 52, 54, 56, 59, and 58
MDa, respectively. Considering the stoichiometry and molar
masses of their structural proteins, the molecular mass of the
protein component of the IBDV virion is 52 ? 2 MDa. In view
of the dsRNA segment sizes (3.2 and 2.8 kbp), and assuming a
molar mass of 682 Da/bp, the molar mass of the IBDV unit
genome component is ?4 MDa.
These analyses support the argument that molar masses of
E2–E6 virions are compatible with the incorporation of an
increasing number of dsRNA molecules, with an average incre-
ment of 1 dsRNA molecule (average molecular mass of ?2
MDa). Our data permit correlation of the relative amount of
nucleic acid for IBDV populations with the number of dsRNA
copies packaged in the virion, and suggest that IBDV popula-
tions correspond to empty particles (E1 fraction) and particles
that package 1 (E2), 2 (E3), 3 (E4), and 4 (E5 and E6) dsRNA
Functional Analysis of IBDV Populations. To determine whether
differences in the packaged genome are related to the specific
infectivity of viral particles [the ratio of physical particles (pp) to
plaque-forming units (pfu)], we titrated the IBDV populations
by plaque assay (Fig. 3). E1 particles were used as a negative
control. The coincident infectivity profiles of E1 and E2 popu-
lations showed residual intrinsic infectivity limited by the puri-
fication of IBDV populations in the same CsCl gradient.
between pfu and pp number for the IBDV populations (Fig. 3).
As a measure of fitness of the virus populations, we calculated
pp/pfu ratios. The values obtained were ?7 ? 103for E1 and E2,
?100 for E3, ?50 for E4, and ?6 for E5 and E6. These results
indicated the molecular basis of the observed phenotype, i.e.,
IBDV populations with a larger number of packed dsRNA
segments were not only functional, but also correlated with
higher infectivity ratios. The data obtained in our experimental
settings suggest that infectivity is a function of nucleic acid
content, but other factors (e.g., the pVP2/VP2 ratio) cannot
strictly be excluded. Accordingly, it appears that the IBDV
of the viral progeny, corresponding to the polyploid virus
population (E5 and E6) (Table S2). These findings suggest that
IBDV particles composed of standard structural proteins in
similar ratios can package multiple genomes to achieve higher
that throughout its evolution, this virus has maintained a much
larger capsid size than is necessary for enclosing a single genome
viruses have been described for which selection pressure has
acted to yield similar consequences.
Viral polyploidy is uncommon among viruses. The only re-
ported polyploid viruses are those with a lipid bilayer, such as
based capsid, and several negative-strand RNA viruses, such as
para- and orthomyxoviruses, which have a helical nucleocapsid
arrangement with total or partial polyploidy, respectively. The
volume enclosed by an icosahedral shell is nonetheless deter-
phoresis and autoradiography of intact (A) or SDS- and proteinase K–treated
protein was analyzed for each IBDV population. vRNA corresponds to viral
to purified dsRNA segments A and B, obtained after SDS and proteinase K
treatments. (C) Relative IBDV dsRNA content based on33P labeling for E2–E6
to the E2 population value. Average and standard deviation values were
calculated from triplicate experiments.
Genome stoichiometry of IBDV populations. Agarose gel electro-
?2-labeled E1–E6 IBDV populations. The same amount of total
Table 2. Biophysical properties of IBDV populations.
290 ? 20
336 ? 12
370 ? 8
418 ? 10
416 ? 15
5.05 ? 0.25
5.47 ? 0.20
5.59 ? 0.15
5.72 ? 0.12
5.75 ? 0.10
52 ? 4
54 ? 2
56 ? 3
59 ? 2
58 ? 2
*Standard sedimentation coefficient averaged over four independent exper-
†dsRNA segment copy numbers compatible with experimental MW. MW of
component and 2 MDa for packaged dsRNA molecule.
E1–E6 populations. Error bars indicate the standard deviations of titrations of
3 independent experiments.
Titration of IBDV populations on QM7 monolayers. Number of
www.pnas.org?cgi?doi?10.1073?pnas.0808498106Luque et al.
mined by capsid geometry, and defines genome size as well as the
copy number of the encapsidated proteins.
Calculations based on structural analyses indicate a high
degree of compaction for dsRNA and dsDNA viruses (1); this is
reflected in an average genome density of ?40 bp/100 nm3
(Table S1). To date, the exception to this rule is represented by
fungal dsRNA virus cores that consistently exhibit a lower
density (?20 bp/100 nm3). These spacious capsids were ex-
plained on the basis of facilitating dsRNA mobility in the
the volume occupied by the ?12 copies of RdRp and the ?450
copies of VP3 (assuming a protein density of 1.3 g/cm3), there is
a clear discrepancy between the available space provided by the
T ? 13 capsid and that occupied by a single copy of the bipartite
genome (Table S1). Packaging of 2 complete genomes or in
general terms, 4 dsRNA molecules (?12 kpb), as determined
experimentally in this study, involves a dsRNA density value
similar to that for dsRNA fungal virus. Even in capsids with a
smaller triangulation number, such as the T ? 7 capsid observed
by heterologous expression of selected IBDV proteins (11),
space would be sufficient to accommodate a single genome copy.
Furthermore, fungal viruses with multipartite dsRNA genomes,
such as chrysovirus (25) and partitivirus (26), package each
dsRNA segment in separate particles rather than in a single
capsid. This feature is also described for comovirus, a group of
plant viruses with a genome consisting of 2 molecules of
positive-strand ssRNA (27).
IBDV segments probably bear specific cis-acting packaging
signals to discriminate between viral and cellular RNA. Random
segment incorporation by highly multipartite genome viruses
(such as reo- and orthomyxoviruses) would lead to a vanishingly
small proportion of fully infectious particles. Thus, whereas
multiple gene segments of influenza virus (28) and bacterio-
phage ?6 (29) are incorporated in a specific order, our data for
IBDV (with only a bipartite genome) provide no evidence of a
mechanism responsible for incorporating these segments in a
given order. This random packaging is supported by the equimo-
lar amounts of segments A and B observed in E2–E6 popula-
molecule, have equal representation of segments A and B.
A random packaging mechanism would involve a pfu/pp ratio
of 0.33 if only 2 dsRNA molecules were incorporated, whereas
the incorporation of 4 molecules would increase 3-fold the
probability of generating an infectious IBDV particle (Fig. S3).
In this context, IBDV polyploidy results in a mechanism that
notably increases the probability of the assembly of virion
particles with a complete functional genome. E4–E6 popula-
tions, which might contain more than 1 complete genome,
account for ?80% of total particles (Table S2); functional
analysis showed a direct correlation between the number of
packaged segments and particle infectivity. Polyploidy also
reduces sensitivity to genetic and environmental perturbation,
i.e., increases robustness.
The molecular differences remain to be established between
E5 and E6 particles, whose infectivity and sedimentation behav-
ior differ slightly. Differences in small single-stranded oligonu-
cleotide content, which forms up to 25% of the RNA in purified
reovirus, may account for a higher sedimentation coefficient for
the E6 population (30). Alternatively, E5 and E6 particles may
have distinct properties, as reported for nodavirus infectious
virions and virus-like particles with cellular RNA, which are
crystallographically identical but have different sensitivities to
The viral replicative cycle of dsRNA viruses is marked by the
ubiquitous presence of a T ? 2 core in which individual mRNA
is transcribed and negative-strand RNA is synthesized from
genome segments that remain enclosed (32). This common,
although not universal, capsid isolates the viral genome from
suppression of gene expression by host responses. Birnaviruses
are a well-established exception and have a single-shelled T ? 13
capsid. Moreover, ribonucleoprotein complexes of birnaviruses
(17) might also represent a functional evolutionary link with
negative-strand ssRNA viruses, in which ribonucleoprotein fil-
aments are common. Furthermore, IBDV RdRP activity is
regulated by the VP3 C-terminal region, which acts as a tran-
scriptional activator; in a similar manner, the polymerase com-
plex of negative-strand RNA viruses can only copy the RNA
sequence in the ribonucleoprotein complex, not naked RNA. It
is currently impossible to identify conserved structural motifs,
since the available VP3 structure lacks the RNA binding region,
precisely where the N proteins from 3 negative-strand ssRNA
virus families show a common topology (33). All these features
together must involve profound differences with regard to the
classical view of the dsRNA virus life cycle. In this scenario,
where there is an excess of packaging capacity, reassortment of
segments A and B—either after coinfection with other viral
strains or, more probably, from naturally occurring variants
during cell infection—might have been a essential mechanism in
Materials and Methods
Purification of Virions. QM7 quail muscle cells were infected with IBDV Soroa
strain at a multiplicity of infection (MOI) of 1–2 pfu per cell. When cytopathic
effect was complete (2 days postinfection), the cell medium was supple-
mented with polyethylene glycol 8000 and NaCl to a final concentration of
3.5% and 0.5 M, respectively. After an incubation (12 h, 4 °C), the virus was
pelleted at 1,000 ? g for 30 min and the resulting pellet was resuspended in
PES buffer [25 mM piperazine-N,N?-bis(2-ethanesulfonic acid) (pH 6.2), 150
(wt/wt) sucrose cushion at 170,000 ? g for 150 min, followed by a CsCl
equilibrium gradient centrifugation for 14 h at 130,000 ? g at 4 °C, adjusting
the initial density of the solution to 1.33 g/ml by addition of CsCl. At least 6
with white light from the bottom, and they were separately collected by side
puncturing. Fractions (?200 ?l) were named E1 to E6 from top to bottom,
Radiolabeling of Infected Cells. QM7 cells were infected with IBDV at an MOI
of 2 pfu/ml. After 1 h of adsorption, the inoculum was replaced with DMEM
supplemented with 2% FCS containing 100 ?Ci/ml [35S]methionine or
protocol described above. [35S]Met- or [33P]HPO4
lyzed by SDS/PAGE, or in 0.7% agarose gels containing 90 mM Tris?HCl (pH 8),
90 mM boric acid, and 20 mM EDTA. To analyze [33P]HPO4
content, E1–E6 samples were incubated with 1% SDS (3 min, 100 °C) and
TriZol (Invitrogen) and purified using the silica-based minispin column (Quia-
gen). Gels were dried on 3MM paper and visualized by phosphorimaging
(Storm 869, Molecular Dynamics). Data were quantitated by using Quantity
the counts in the bands were measured and normalized considering the
number of Met residues (21, 9, 9, and 6 for VP1, (p)VP2, VP3, and VP4,
respectively; the C-terminal region of pVP2 that is processed during virus
maturation has no Met residues). The copy numbers of VP1, VP3, and VP4
molecules were normalized to the internal standard given by the 780 copies
of VP2 and pVP2, as unequivocally established by X-ray crystallography and
E2–E6 populations was determined in each35S-labeling experiment. Data are
averages from triplicate experiments.
?2, respectively. Cell medium of IBDV-labeled virions was mixed with
?2-labeled virions were ana-
Electron Microscopy of IBDV Particles. Samples (?5 ?l) were applied to glow-
discharged carbon-coated grids for 2 min. Samples were stained with 2%
aqueous uranyl acetate. Micrographs were recorded with a JEOL 1200 EXII
electron microscope operating at 100 kV at a nominal magnification of
Biophysical Analysis of E2–E6 IBDV Population Particles. Sedimentation velocity.
Experiments were done using IBDV particles E2–E6 (at 0.1–0.5 mg/ml) equil-
ibrated in PES buffer. Sedimentation velocity runs were carried out at 10,000
Luque et al.PNAS ?
February 17, 2009 ?
vol. 106 ?
no. 7 ?
rpm and 20 °C in an XLA-analytical ultracentrifuge (Beckman-Coulter) using Download full-text
an An50Ti rotor and 12-mm double-sector centerpieces, and measuring ab-
sorbance at the appropriate wavelength. Sedimentation coefficient distribu-
tion was calculated by least-squares boundary modeling of sedimentation
velocity data using the c(s) method (34). These s-values were corrected to
standard conditions [water, 20 °C, and infinite dilution (23)] to obtain the
corresponding standard s-values (s20,w).
Dynamic light scattering (DLS). DLS experiments were conducted with a Protein
Solutions DynaPro-MS/X instrument. Buffers were filtered by a 100-nm filter
dust particles. Samples (20 ?l) were inserted in the 90° light scattering cuvette
at 20 °C. Diffusion coefficients of the scattering solute particles (assuming a
globular shape of the protein species) were calculated using the SEDFIT
program adapted for DLS by replacing the single species Lamm equation
were corrected for buffer and concentration (23) to get the standard D0
coefficients. Molecular masses of E2–E6 IBDV particles were calculated by the
(106cells per assay). Briefly, virus stocks were serially diluted with serum-free
in DMEM supplemented with 2% FCS was added to each plate. Cells were
inoculated in triplicate with each virus dilution. After 48 h, cells were fixed in
10% formaldehyde and stained with 2% crystal violet. For gradient-purified
E1–E6 band IBDV populations, the amount of total protein was determined
using the BCA protein assay kit (Pierce), and infections were performed at the
same protein concentration for each IBDV population.
ACKNOWLEDGMENTS. We thank C. Mark for editorial help. This work was
supported by grants BFU 2005–06487 and BIO 2006–09407 from the Spanish
Direccio ´n General de Investigacio ´n (MEC).
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