A single mutation in the PBC loop of VP2 is involved in the in vitro replication of infectious bursal disease virus
A single mutation in the PBC loop of VP2 is involved in the in vitro replication of infectious bursal disease virus
Xiaole Qi 0 2 3
Xiang Gao 0 2 3
Zhen Lu 0 2 3
Lizhou Zhang 0 2 3
Yongqiang Wang 0 2 3
Li Gao 0 2 3
Yulong Gao 0 2 3
Kai Li 0 2 3
Honglei Gao 0 2 3
Changjun Liu 0 2 3
Hongyu Cui 0 2 3
Yanping Zhang 0 2 3
Xiaomei Wang 0 1 2 3
0 Qi , X., Gao, X., Lu, Z., Zhang, L., Wang, Y., Gao, L., Gao, Y., Li, K., Gao, H., Liu, C., Cui, H., Zhang, Y., and Wang, X. (2016). A single mutation in the P
1 Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Disease and Zoonoses , Yangzhou 225009 , China
2 infectious bursal disease virus (IBDV) , VP2, P
3 Division of Avian Infectious Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences , Harbin 150001 , China
To test whether amino acid mutations in the PBC and PHI loops of VP2 are involved in the replication and virulence of infectious bursal disease virus (IBDV), a pair of viruses, namely the moderately virulent IBDV (rGx-F9VP2) and the attenuated strain (rGt), were used. Residue mutations A222P (PBC) and S330R (PHI), selected by sequence comparison, were introduced individually into rGx-F9VP2 by using a reverse genetics system. In addition, the reverse mutation of either P222A or R330S was introduced into rGt. The four modified viruses were then rescued and evaluated in vitro (CEF cells) and in vivo (SPF chickens). Results showed that A222P elevated the replication efficiency of rGx-F9VP2 while P222A reduced that of rGt in CEF cells. A mutation at residue 330 did not alter IBDV replication. In addition, animal experiments showed that a single mutation at either residue 222 or 330 did not significantly influence the virulence of IBDV. In conclusion, residue 222 in PBC of VP2 is involved in the replication efficiency of IBDV in vitro but does not affect its virulence in vivo, further facilitating our understanding of the gene-function of IBDV.
Infectious bursal disease virus (IBDV) is the causative agent
of a highly contagious disease in chickens known as
infectious bursal disease (IBD). IBD causes significant losses of
the poultry industry owing to its high mortality and
immunosuppression in young chickens via the destruction of
developing B lymphocytes in the bursa (Cosgrove, 1962;
Muller et al., 2003). IBDV belongs to the Birnaviridae
family and has a genome that consists of two segments of
double-stranded RNA (segments A and B). Segment B encodes
the VP1 protein, the viral RNA-dependent RNA polymerase
(Le Nouen et al., 2006; von Einem et al., 2004), while
segment A contains two partially overlapping open reading
frames (ORF). The smaller ORF encodes the non-structural
protein VP5 (Mundt et al., 1995), while the larger one
encodes a polyprotein which is cleaved into proteins pVP2,
VP3, and VP4 (Birghan et al., 2000). VP2 and four peptides
are further derived from the maturation of pVP2 (Da Costa
et al., 2002).
As the sole component of the icosahedral capsid, VP2 is
folded into three distinct domains, designated the base (B),
shell (S), and projection (P) (Coulibaly et al., 2005; Garriga
et al., 2006; Lee et al., 2006). Domains B and S are
relatively well conserved, whereas domain P is more variable.
The tower-like P domain contains four loops, PBC, PHI, PDE,
© The Author(s) 2016. This article is published with open access at link.springer.com
and PFG. It has been reported that the PDE and PFG domains
are responsible for cell-tropism and virulence (Brandt et al.,
2001; Qi et al., 2009, 2013; van Loon et al., 2002). The
other two loops, PBC and PHI, are also located at the tip of the
VP2 spike; however, their roles in viral replication and
virulence are not fully understood.
Previously, a moderately virulent IBDV strain
(rGx-F9VP2) and an attenuated strain (rGt) were rescued,
providing a good model system for studying the viral
replication and virulence of IBDV (Qi et al., 2007, 2009, 2013).
In the current study, specific amino acid mutations in the
PBC and PHI domains of VP2 were introduced into the
backbone of the virulent (rGx-F9VP2) and attenuated (rGt)
strains in order to evaluate the roles of these amino acid
changes in replication and virulence.
Sequence analysis of the PBC and PHI domains of VP2
Sequence alignment showed that, compared to virulent
IBDV including rGx-F9VP2, two amino acids mutations,
one at loop PBC (A222P) and another at loop PHI (S330R),
were observed in the attenuated strain including Gt
Rescue and identification of the modified IBDV
In cells co-transfected with pCGxATA-G794CHRT/
pCGxBHRT or pCGxATA-T1120AHRT/pCGxBHRT, the
modified IBDV rGxHT-222 or rGxHT-330 strains were
rescued, which contained a single mutation A222P in PBC or
S330R in PHI of VP2 compared to the virulent strain
rGxF9VP2, respectively. In cells co-transfected with
pCmGtA-C794GHRT/pCmGtBHRT or pCmGtA-A1120THRT/
pCmGtBHRT, the modified IBDV rGt-222 or rGt-330 strain
was rescued, which contained the reverse mutation P222A
or R330S compared to the attenuated strain rGt.
Replication characteristics of the modified IBDV
To investigate the replication character in vitro of the
modified IBDV strains in detail, a replication kinetics
curve in CEF cells was depicted (Figure 2). Compared to
the parental strain of rGx-F9VP2, rGxHT-222 replicated
more efficiently, where the titers were significantly higher
than rGx-F9VP2 after 36 h post-infection (p.i.) (P<0.05).
rGxHT-330 showed a similar curve to rGx-F9VP2 (Figure
2A). Compared to the rGt parental strain, rGt-222 replicated
less efficiently in CEF cells after 48 h p.i.. The titer of
rGt-222 was 106.7 TCID50 mL−1 at 60 h p.i., which was ten
times lower than that of rGt (P<0.05) (Figure 2B). rGt-330
showed a similar curve to rGt (Figure 2B).
The pathogenicity of the modified IBDV to chicken
The pathogenicity of the modified IBDV rGxHT-222 and
rGxHT-330 strains was evaluated using three-week-old SPF
chickens. Neither death nor clinical symptoms of IBD were
observed in any of the groups during the experimental
period. In order to investigate the underlying sub-clinical lesion,
the BBIX at different days p.i. was calculated. The BBIX of
the rGxHT-330 and rGx-F9VP2 groups were below 0.7
from 3 d p.i. to 14 d p.i., indicating signs of atrophy. In the
rGxHT-222 group, a BBIX above 0.7 was observed before
3 d p.i. and then the BBIX decreased to below 0.7 (Figure
The histopathological changes of the infected bursae are
presented in Figure 3B. Chickens from all groups,
rGxHT-222, rGxHT-330, and rGx-F9VP2, showed
persistent histopathological bursal lesions, including lymphocytic
deletion and necrosis, regional atrophy, and fibrosis of the
follicle (average HBLS was 4). No obvious bursal lesions
were observed in the DMEM control group (HBLS was 0).
RT-PCR and sequencing results confirmed that the
modified viruses replicated in the bursae of chickens without
causing additional mutations (data not shown).
305 330 337
Figure 1 Comparison of the amino acid sequences of the PBC (aa 204–237) and PHI domain (aa 305–337) of IBDV VP2 from virulent and attenuated strains.
The amino acids differences are marked.
Figure 2 Replication characteristics of the modified IBDV. Replication
kinetics curves in vitro of the modified IBDV based on backbone of
rGx-F9VP2 and rGt strains are shown in (A) and (B), respectively.
Average titers and standard deviations (error bars) from three independent
samples are shown. Treatments sharing different lowercase letters differ
significantly at a confidence level (P<0.05).
As an RNA virus, IBDV is genetically prone to mutation. In
terms of disease control and early warning, it is important to
fully understand the gene or residue function. Since the
development of reverse genetics, the role of VP2 in replication
or virulence has attracted much attention (Brandt et al.,
2001; Li et al., 2015; Qi et al., 2009, 2013; van Loon et al.,
2002). However, the precise molecular determinants are still
somewhat unclear. The synergistic effects of multi-genes or
multi-residues are indispensable (Boot et al., 2000; Le
Nouen et al., 2006), although the virulence and replication
of many viruses, including IBDV, mostly depend on the
major corresponding gene. The four loops of VP2, PBC, PHI,
PDE, and PFG, are located at the outermost surface of IBDV.
Loops PDE and PFG influence cell-tropism and virulence and
have been researched in detail (Brandt et al., 2001; Lim et
al., 1999; Mundt, 1999; Qi et al., 2009, 2013; van Loon et
al., 2002). While the PBC and PHI residues are unlikely to be
involved in three-dimensional folding (Letzel et al., 2007),
mutations at some residues mutations are thought to directly
or indirectly alter the characteristics of VP2 via different
mechanisms (Durairaj et al., 2011; Letzel et al., 2007).
Therefore, we hypothesized that PBC and PHI might
contribute to viral replication or virulence.
Sequence analysis showed that two conserved residue
differences, A222P (PBC) and S330R (PHI), were observed in
Figure 3 Evaluation of the influence of residue 222 or 330 of VP2 on the virulence of IBDV. A, Kinetics curves of bursa:body-weight index (BBIX) of
SPF chickens infected with the modified virus. Average titers and standard deviations (error bars) from three independent samples are shown. Treatments
sharing different lowercase letters differ significantly at a confidence level (P<0.05). B, Histopathological appearance of bursal sections (hematoxylin and
eosin) derived from groups of chickens infected with the modified viruses.
the PBC and PHI domains between virulent and attenuated
IBDV strains. The virulent IBDV strain rGx-F9VP2 and
attenuated strain rGt were chosen to verify the hypothesis
because they have a similar gene background but have
different characters. Firstly, A222P (PBC) and S330R (PHI)
were introduced into the backbone of rGx-F9VP2, and the
resulting mutated viruses rGxHT-222 and rGxHT-330 were
rescued. rGt replicated more efficiently in vitro compared
with rGx-F9VP2. Replication of the mutated viruses was
then evaluated. In CEF cells, it was clear that A222P of PBC
could significantly increase the replication of rGx-F9VP2
while S330R of PHI could not. To further verify these
results, the reverse mutation P222A or R330S was introduced
into rGt and the rescued virus rGt-222 replicated less
efficiently than rGt, while rGt-330 showed similar replicate
curve to rGt. The data obtained from these different
backbones verified that residue 222 of PBC in VP2 could
definitely influence the replication efficiency of IBDV in vitro
but that a mutation in residue 330 could not.
Residue 222 of VP2 is typically A in vvIBDV and P in
non-vvIBDV strains. Sometimes, T residue at position 222
of VP2 has also been observed in a few variant strains,
including DEL-E, GLS, and GA988 (Jackwood and
Sommer-Wagner, 2011; Letzel et al., 2007). Recently, a
new occurring mutation P222S was observed in Belg strain
(Letzel et al., 2007). A single substitution at position 222
(P222S or A222T) was able to alter the antigenic pattern of
IBDV because an S or T residue at position 222 is the
essential part of the epitope that reacts with MAb 67 (Letzel et
al., 2007). Recently, a natural mutation T222A was shown
to allow the Del-E strain to break through the maternal
immunity induced by parenteral vaccination (Jackwood and
Residue 330 belongs to the heptapeptide-domain (aa
326-332) that closely follows the major hydrophilic peak B
(aa 312-324 of VP2). There is usually a conserved amino
acid difference at residue 330, which is R in attenuated and
S in non-attenuated strains. Residue 330 had originally been
thought to influence cell-tropism of IBDV; however, this
has since been refuted (Brandt et al., 2001; Lim et al., 1999;
Mundt, 1999). Recently, it was reported that recognition of
the MAb 57 epitope involves the PHI loop, and specifically a
residue at position 330 (Letzel et al., 2007). The
threedimensional structure shows that residue 330 is located at
the interface between subunits in the trimeric spike of VP2
and a mutation here could lead to subtle alterations in the
jelly roll, ultimately leading to a different conformation of
loop PBC (Letzel et al., 2007). It is possible that the subtle
change in the conformation of PBC induced by the residue
330 mutation is not sufficient to alter the assembly and
stability of the virus such that S330R cannot influence the
replication of IBDV.
The virulence of rGxHT-222 and rGxHT-330 was further
evaluated using SPF chickens. As was reported for
rGxF9VP2, chickens in both the rGxHT-222 and rGxHT-330
groups did not present any typical clinical symptoms. To
further detect the pathological lesions, the BBIX and the
degree of damage to the bursae were evaluated, parameters
which have been used in many studies of sub-clinical
disease (Boot et al., 2000; Brandt et al., 2001; Jackwood et al.,
2008; Le Nouen et al., 2006; Liu and Vakharia, 2004; Qi et
al., 2009; Raue et al., 2004; Rautenschlein et al., 2003).
Atrophy of the bursa was not found in chicken samples
from the rGxHT-222 group at 3 d p.i., but rGxHT-222,
rGxHT-330, and rGx-F9VP2 did cause obvious
histopathological bursal lesions. There is typically a negative
correlation between the replication efficiency in cell culture and
the virulence of IBDV. However, data from the current
study demonstrate that the A222P mutation elevated the
viral replication efficiency in vivo but did not obviously
attenuate rGx-F9VP2. Further studies will be required to
uncover the molecular mechanism that underlies this effect.
In conclusion, the current study represents the first
demonstration that residue 222 in the PBC loop of VP2 is
involved in the replication efficiency of IBDV in vitro but does
not have a role in its virulence in vivo. A mutation at residue
330 in the PHI loop of VP2 did not contribute to the
replication and virulence of IBDV. These findings are beneficial to
our further understanding of the gene-function of IBDV.
MATERIALS AND METHODS
Viruses, cells, and plasmids
The rGx-F9VP2 strain of IBDV, which shows moderate
virulence, was previously rescued and was shown to induce
severe bursal lesions in chickens but did not cause mortality
(Qi et al., 2009). The rGt virus was previously rescued from
the infectious clones of the attenuated strain of Gt and has
similar characteristics to the parental strain (Qi et al., 2007).
DF-1 cells were cultured in Dulbecco’s modified eagle
medium (DMEM) (Invitrogen, USA) supplemented with 10%
fetal bovine serum at 37°C in a humidified 5% CO2
incubator. Chicken embryo fibroblast (CEF) cells were prepared
from 10-day-old specific-pathogen-free (SPF) chicken
embryos. The infectious clones pCmGtAHRT and
pCmGtBHRT (containing segments A and B of rGt) (Qi et al.,
2007), and the infectious clones pCGxAF9VP2HRT and
pCGxBHRT (containing segments A and B of rGx-F9VP2)
were constructed previously (Qi et al., 2009).
SPF chickens were purchased from the Experimental
Animal Center of the Harbin Veterinary Research Institute
(HVRI), the Chinese Academy of Agricultural Sciences
(CAAS), and were housed in negative-pressure-filtered air
isolators. All animal experiments were approved by the
Animal Ethics Committee of HVRI.
Sequence alignment of the PBC and PHI domains of VP2
To identify interesting amino acid residue differences, the
amino acid sequences of the PBC (aa 204 to 237) and PHI (aa
305 to 337) domains of VP2 of virulent and attenuated
strains were compared. Virulent strains: D6948 (GenBank
accession no. AF240686), HK46 (AF092943), UK661
(X92760), OKYM (D49706), KS (DQ927042), Gx
(AY444873), HuB-1(KF569805), SD10LY01 (KF569803),
T09 (AY099456). Attenuated strains: CU1 (X16107), D78
(AF499929), P2 (X84034), CT (AJ310185), Gt
(DQ403248). Alignment based on the amino acid sequences was
performed using DNAStar (5.01 edition).
Table 1 Primers for genome cloning and mutagenesis of IBDV.
Modification of segment A of IBDV using site-directed
To introduce direct mutations into segment A of the
rGxF9VP2 strain, based on the parental pCGxAF9VP2HRT
plasmid, PCR for site-directed mutagenesis with specific
primer pairs was performed as described previously (Qi et
al., 2007). The primer pair GxAG794CU/GxAG794CL or
GxAT1120AU/GxAT1120AL (Table 1) was synthesized to
introduce a direct mutation G794C or T1120A (resulting
in an amino acid mutation A222P or S330R in VP2) into
segment A of the rGx-F9VP2 strain. The mutated
plasmid was named pCGxATA-G794CHRT and
pCGxATA-T1120AHRT, respectively (Figure 4A). Similarly, to
Figure 4 Schematic diagrams of the infectious clones containing the modified cDNAs of segment A of IBDV (not drawn to scale). Mutations were
introduced into VP2 of segment A backbone of the virulent strain rGx-F9VP2 (depicted by an open box) (A) or the attenuated strain rGt (depicted by a box with
dot) (B). The restriction enzyme sites used for the construction of recombinant vectors are also shown.
introduce a reverse mutation into segment A of the rGt
strain, based on the parental plasmid pCmGtAHRT, a
primer pair GtAC794GU/GtAC794GL or
GtAA1120TU/GtAA1120TL (Table 1) was used to introduce
a nucleotide mutation C794G or A1120T (resulting in
an amino acid mutation P222A or R330S in VP2) into
segment A of rGt to obtain the modified plasmids
pCmGtA-C794GHRT and pCmGtA-A1120THRT,
respectively (Figure 4B).
Rescue and identification of modified IBDV
Using the reverse genetics system directed by RNA
polymerase II, virus rescue was performed as previously
described (Qi et al., 2007). The purified plasmids with the
rGx-F9VP2 backbone (pCGxATA-G794CHRT or
pCGxATA-T1120AHRT) were co-transfected with pCGxBHRT
into DF-1 cells. The purified plasmids with the rGt
backbone (pCmGtA-C794GHRT or pCmGtA-A1120THRT)
were co-transfected with pCmGtBHRT into DF-1 cells.
Three days post-transfection, the cell cultures were
freezethawed three times and the supernatants were transferred to
fresh CEF cells. The viruses were harvested from the
infected cell cultures when a visible cytopathic effect (CPE)
was apparent. The modified viruses were blind-passaged six
times in CEF cells prior to subsequent experiments. To
characterize the modified viruses, an indirect
immunofluorescence assay (IFA) with an anti-VP2 mAb, an electron
microscopy assay, RT-PCR, and sequencing using the
primer pairs GxAU/GxA1477L and B3P/B37 (Table 1)
were performed as previously described (Qi et al., 2007).
Replication of modified IBDV in vitro
To assess the replication abilities of the modified viruses
and the control strain rGx-F9VP2 or rGt, confluent
secondary CEF cells in 60 mm culture plates (approximately 106
cells/plate) were infected with each virus strain at a 50%
cell culture infective dose (TCID50) of 1×104 and were
subsequently harvested at 24, 36, 48, 60, and 72 h p.i.. The titer
of the infectious viral progeny was determined as TCID50
per milliliter using the Reed-Muench formula (Reed and
Muench, 1938). The mean values and standard deviations of
the data obtained from three independent experiments were
Three-week-old SPF chickens were randomly divided into
four groups (eighteen chickens per group). Chickens were
inoculated via ocular and intranasal routes with 105.8
TCID50 of rGxHT-222, rGxHT-330, and rGX-F9VP2.
DMEM was used as a negative control. Chickens were
observed daily for signs of clinical symptoms. At 2, 3, 5, 7,
10, and 14 d p.i., three chickens were randomly selected
from each group, euthanized for necropsy, and examined
for signs of pathological changes. The bursa:body-weight
index (BBIX) was calculated with standard deviation
ratios in the negative group)). The mean values and standard
deviations of the data obtained from three independent
chicken samples were calculated. Bursa with a BBIX lower
than 0.7 were considered atrophied (Lucio and Hitchner,
1979). Each bursa was then divided into two parts, one for a
histopathological assay and the other for detecting the viral
Bursae from each group isolated on different days p.i. were
fixed immediately after necropsy in 10% neutral buffered
formalin and were stained with hematoxylin and eosin for
histopathological examination, as described previously (Qi
et al., 2009). The severity of bursal follicular necrosis was
recorded using the average histopathologic bursa lesion
score (HBLS) system, as described earlier (Schroder et al.,
One-way ANOVA was used to evaluate the significant
differences among the different groups. P<0.05 was
considered a significant difference.
Acknowledgements This work was supported by the National Natural
Science Foundation of China (31430087), the Scientific and Technological
Research Project of Harbin (2014AB3AN058), the Special Fund for
Scientific and Technological Innovative Talents of Harbin (2014RFQYJ129),
and the Modern Agro-industry Technology Research System of China
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