Construction of a full-length infectious bacterial artificial chromosome clone of duck enteritis virus vaccine strain
Construction of a full-length infectious bacterial artificial chromosome clone of duck enteritis virus vaccine strain
Liu Chen 0
Bin Yu 0
Jionggang Hua 0
Weicheng Ye 0
Zheng Ni 0
Tao Yun 0
Xiaohui Deng 0
Cun Zhang 0
0 Institute of Animal Husbandry and Veterinary Medicine, Zhejiang Academy of Agricultural Sciences , Hangzhou 310021 , China
Background: Duck enteritis virus (DEV) is the causative agent of duck viral enteritis, which causes an acute, contagious and lethal disease of many species of waterfowl within the order Anseriformes. In recent years, two laboratories have reported on the successful construction of DEV infectious clones in viral vectors to express exogenous genes. The clones obtained were either created with deletion of viral genes and based on highly virulent strains or were constructed using a traditional overlapping fosmid DNA system. Here, we report the construction of a full-length infectious clone of DEV vaccine strain that was cloned into a bacterial artificial chromosome (BAC). Methods: A mini-F vector as a BAC that allows the maintenance of large circular DNA in E. coli was introduced into the intergenic region between UL15B and UL18 of a DEV vaccine strain by homologous recombination in chicken embryoblasts (CEFs). Then, the full-length DEV clone pDEV-vac was obtained by electroporating circular viral replication intermediates containing the mini-F sequence into E. coli DH10B and identified by enzyme digestion and sequencing. The infectivity of the pDEV-vac was validated by DEV reconstitution from CEFs transfected with pDEV-vac. The reconstructed virus without mini-F vector sequence was also rescued by co-transfecting the Cre recombinase expression plasmid pCAGGS-NLS/Cre and pDEV-vac into CEF cultures. Finally, the in vitro growth properties and immunoprotection capacity in ducks of the reconstructed viruses were also determined and compared with the parental virus. Results: The full genome of the DEV vaccine strain was successfully cloned into the BAC, and this BAC clone was infectious. The in vitro growth properties of these reconstructions were very similar to parental DEV, and ducks immunized with these viruses acquired protection against virulent DEV challenge. Conclusions: DEV vaccine virus was cloned as an infectious bacterial artificial chromosome maintaining full-length genome without any deletions or destruction of the viral coding sequence, and the viruses rescued from the DEV-BAC clone exhibited wild-type phenotypes both in vitro and in vivo. The generated infectious clone will greatly facilitate studies on the individual genes of DEV and applications in gene deletion or live vector vaccines.
Duck enteritis virus; Bacterial artificial chromosome; Infectious clone
Duck virus enteritis (DVE), also known as duck plague,
is an acute contagious infection of ducks, Muscovy ducks,
geese, and swans (order Anseriformes) that is caused by
duck enteritis virus (DEV). According to the most recent
virus taxonomy reported in 2012 by the International
Committee on Taxonomy of Viruses (ICTV), DEV (also
referred to as Anatid herpesvirus 1) is classified into the
genus Mardivirus and the subfamily Alphaherpesvirinae
of Herpesviridae . This virus can infect ducks of both
sexes of a wide age and has high morbidity and mortality.
The disease is characterized by extensive hemorrhaging
and necrosis, most commonly in the digestive and
lymphoid organs. DVE has resulted in significant economic
losses in domestic and wild waterfowls worldwide. At
present, the major approaches to preventing and
controlling lethal DEV infections in ducks is to inoculate
ducks with live attenuated DEV vaccines.
Herpesvirus is a large, enveloped virus with four
structural components, including linear double-stranded
DNA, an icosahedral capsid, an amorphous tegument,
and a bilayer lipid envelope. The genomes of these
viruses differ in size, sequence arrangements, and base
composition, and they also vary significantly with respect
to the presence and arrangement of inverted and directly
repeated sequences [2,3]. Partial or complete genomic
sequences of DEV have been obtained, and there has
been discrepancies found between these sequences, which
demonstrate that although similar to other herpesviruses,
the DEV genome also varies [4-10]. In general, the DEV
genome is approximately 158 kb and contains 78 ORFs
predicted to encode potential functional proteins. In
addition, the genome also contains a unique long (UL)
region, a unique short (US) region, a unique short internal
repeat (IR) region, and a unique short terminal repeat
Bacterial artificial chromosomes (BACs) have proven
to be useful vectors for cloning large gene fragments,
including viral genomes. A combination of a viral BAC
system and various Escherichia coli-based recombination
systems is a highly reliable and efficient method for
generating a variety of different modifications of BAC
clones, which are fundamental tools for applications as
diverse as the elucidation of viral gene or domain function,
the generation of transgenic animals, and the construction
of gene therapy or vaccine vectors [11-13]. The genomes
of many herpesviruses, including cytomegalovirus (CMV),
Marek’s disease virus (MDV), koi herpesvirus (KHV),
varicella zoster virus (VZV), bovine herpesvirus type 1
(BoHV-1), equine herpesvirus 4 (EHV-4), and
pseudorabies virus (PRV), have been cloned as bacterial artificial
chromosomes in E. coli [14-20]. The infectious viral
BAC of DEV virulent strain 2085 was first constructed
in 2011 by Wang et al. . Then, Liu et al. established
a system to generate DEV vaccine strain by transfecting
overlapping fosmid DNAs . Here, we report the
construction of an infectious clone of a Chinese DEV
vaccine C-KCE strain that has been commercialized
and widely used in preventing DEV infection in China.
This DEV BAC system will facilitate the studying of DEV
biology and gene functions as well as novel vaccines.
Generation of recombinant DEV harboring mini-F
CEFs were transfected with the linearized transfer vector
pHA2-UL18-UL15 and then infected with DEV vaccine
strain (DEV-vac). Drug screening, combining plaque
picking and passaging of end-point dilutions of
recombinant progeny virus identified by GFP expression, were
performed. Purified recombinant rDEV, which carries
miniF sequences inserted within the non-encoding region
between UL18 and UL15B, was obtained after 10 rounds
of plaque and end-point purification (Figure 1).
Generation and validation of a supercoiled
Circular viral DNA was isolated from rDEV-infected cells
by the SDS-proteinase K method and transferred into
E. coli by electroporation. Several
chloramphenicolresistant colonies were obtained 36 h after plating of
electroporated cells, and RFLPs (restriction fragment length
polymorphisms) were determined to confirm that a
full-length DEV-vac BAC clone was indeed generated.
The DNA of two selected colonies was isolated and
digested with four enzymes: EcoR V, Bgl II, BamH I,
and Xbal I. The electrophoresis results indicated that
the bands from two colonies were completely identical.
When the RFLP patterns were compared to in silico
predictions, which were based on the reference whole
genome sequence of the DEV vaccine strain [GenBank:
KF487736] , the obtained BamH I pattern matched
those of the predictions well, and the other enzyme
digestion patterns displayed a few differences from the
predictions (Figure 2).
Nucleotide sequence of pDEV-vac
To further evaluate the integrity of the generated
infectious clones, whole genome sequencing of pDEV-vac
[GenBank: KF693236] and comparison with the reference
sequence [GenBank: KF487736] was performed. The
results of the whole genome sequencing of pDEV-vac
revealed that there were only four nucleotide changes
in the viral coding sequence: a silent mutation in UL36, an
H1114 P mutation in UL36, an I205V mutation in UL23,
and an R185Q mutation in UL3. There were seven site
changes, including two 80-nt repetitive sequence deletions
in the non-coding region, compared with the published
sequence (Table 1).
To construct an infectious DEV clone using a BAC
system, the process, which successively includes “virus
Figure 1 Homogeneous population of rDEV in CEFs (100×). A.
Fluorescence under UV excitation; B. Phase contrast.
Figure 2 Restriction fragment analysis of full-length BAC clone pDEV-vac. A. The patterns corresponded exactly to the predictions based on
the complete DEV genome (KF487736), as shown by in silico digests using Vector NTI. B. BAC DNA was isolated and digested with four different
enzymes (EcoR V, Bgl II, BamH I, and Xbal I) and separated with a 1.0% agarose gel. The sizes of a molecular weight marker (15,000-bp marker,
Takara) are given.
passage and recombination in cells”, “from recombinant
virus to BAC” (that is, from cells to bacteria), and “from
BAC to virus” (that is, from bacteria back to cells) can
potentially lead to changes in the viral genome. To further
illuminate in which step these mutations were introduced,
we sequenced 11 sites in four virus (DEV-vac, rDEV,
rDEV-BAC, and rDEV-Cre) genomes and compared them
with pDEV-vac and the reference sequence [GenBank:
KF487736]. The results are shown in Table 2. The results
indicated that the majority of changes occurred in the
“virus passage and recombination in cells” step,
including A41128G, A43596C, A75096G, and C110795T. A minor
mutation occurred in the “from cells to bacteria” step,
e.g., an 80-nt deletion in 119773–119852. Some changes
occurred in both the “virus passage and recombination
in cells” and “from cells to bacteria” steps, including
changes in sites 129421–129422, 147986–147987, and
157498–157877. In addition, no mutations occurred in
the “from bacteria back to cells” step. T59643, T143005 and
G158090 were the same as in the parental virus DEV-vac.
Reconstitution and characterization of BAC-derived DEV
pDEV-vac DNA was transfected into CEFs by calcium
phosphate precipitation to produce the recombinant
virus rDEV-BAC. A typical cytopathic effect (CPE) with
green fluorescence was observed in the cell monolayer
4 days later. To exclude the effect of additional BAC
sequences on the virus, the BAC sequence, including the
xgpt and gfp genes, was excised by site-specific
recombination using flanking loxP sites. This excision
was achieved by co-transfecting the Cre recombinase
expression plasmid pCAGGS-NLS/Cre and pDEV-vac
into the CEF cultures. Following this step, a typical
DEV CPE without green fluorescence appeared; then,
the cloned virus without the BAC sequence, named
rDEV-Cre, was obtained by limiting dilution and plaque
purification. The correct excision of BAC sequences from
the recombinant virus rDEV-Cre was confirmed by PCR
and sequencing (data not shown).
To compare the growth of the recombinant virus and
the parental virus, the multi-step growth kinetics of the
reconstructed viruses were determined and compared with
DEV-vac. As shown in Figure 3, all reconstituted viruses,
rDEV, rDEV-BAC, and rDEV-Cre (both intracellular
and extracellular), exhibited growth characteristics that
were virtually identical to each other and to those of
parental DEV-vac. In addition, the virus titers steadily
increased from 12 to 60 h post-infection. When the
plaque areas of the reconstructed viruses rDEV,
rDEVBAC, and rDEV-Cre were separately compared to parental
virus DEV-vac, it was discovered that the plaque areas
were approximately 7.63%, 10.38%, and 4.16% smaller,
Table 1 List of nucleotide sequence different between
cloned pDEV-vac [GenBank: KF693236] and the published
DEV vaccine sequence [GenBank: KF487736]
A. Changes in coding sequences
A to C (43 596)
A to G (75 096)
Deletion 80 nt : GGGTTCCAAAGGTTTTACGGTA
(119 773–119 852)
Insertion: AA (129 421–129 422)
T to G (143 005)
Insertion: TT (147 986–147 987)
Deletion 80 nt:
AACCCTGCCAACCCTAAC (157 498–157 877)
Insertion: G (158 090)
Between IR and US1
Between US1 and TR
respectively, than those formed by DEV-vac when
measured on day 4 p.i., but there were no significant differences
between the groups (P = 0.241; P = 0.05; P = 0.485), and
there was also no significant difference between rDEV and
rDEV-BAC (P = 0.554) (Figure 4). These data demonstrate
that the insertion of mini-F sequence into DEV’s genome
and/or proliferation of viral genome in E. coli had a slight
effect on the viral plaque area.
Reconstructed virus could protect ducks against virulent
To investigate whether the reconstructed viruses could
protect ducks against virulent DEV challenge, the ducks
were first inoculated with 1 × 105 TCID50 of DEV-vac,
rDEV, rDEV-BAC, rDEV-Cre, or with culture medium as
a control for 2 weeks and then challenged with highly
virulent DEV. Four days later, the clinical symptoms of
duck viral enteritis and death of the ducks were observed
only in the negative control groups, and all ducks in the
control groups died within 8 days post-challenge. The
other groups, which had been previously inoculated
with reconstructed viruses or DEV-vac, remained healthy
during the 2-week observation period. The survival rates
of the different groups are summarized in Figure 5.
Tissues from all birds were also examined for
pathological lesions. The gross lesions were characterized by
vascular damage, with tissue hemorrhages, especially
in liver, spleen, esophagus, and cloaca, and there was
annular band necrosis and hemorrhage in the intestinal
tract, along with diphtheroid lesions in the mucosal
surfaces of the esophagus and cloaca.
Although DEV is an important pathogen in waterfowl
that causes high morbidity and mortality in infected
birds, little is known about the function of viral genes
and proteins, the molecular and cellular mechanisms of
different phases of the DEV life cycle, and the mechanisms
involved in DEV virulence and pathogenicity. Full-length
genomic clones from DEV strains allowing the recovery
of infectious virus would be an invaluable tool for various
in-depth investigations on these questions. To date, there
have been only two reports detailing the construction
of DEV infectious clones, and based on the established
platform, exogenous genes such as the H5N1 HA gene
were successfully expressed. In one case, the recombinant
virus could provide fast and complete protection against
H5N1 avian influenza virus infection in ducks [21,22].
These results demonstrate that DEV is a potential viral
Compared with an overlapping fosmid DNA system,
there are more advantages to cloning the viral genome
into a BAC system, as it allows stable maintenance the
viral genome in E. coli and also allows a variety of different
modifications to the viral genome to be easily generated
using various E. coli-based recombination systems. It
has been reported that the insertion of foreign genes
into the intergenic region between genes UL15 and
UL18 in BoHV was stable and had no effect on viral
growth in cell culture . In this study, the same site
was chosen for construction of infectious viral BAC for
the DEV vaccine strain. This infectious DEV clone was
successfully constructed by inserting the bacterial mini-F
plasmid sequences flanked by loxP sites into the intergenic
region between genes UL15B and UL18 of DEV without
deletion of any viral sequence through homologous
recombination. Our results demonstrate that exogenous
sequence insertion between the UL15B and UL18 genes
had no effect on DEV replication. The plaque area
measurement and multi-step growth kinetics results reveal that
insertion of mini-F sequence into DEV’s genome and/or
proliferation of the viral genome in E. coli had a slight
effect on virus transmission during cell-to-cell spread but
did not affect the virus growth pattern in vitro.
Furthermore, these processes did not increase the virulence of the
Table 2 Comparasion of mutated sites in pDEV-vac (KF693236), the published DEV vaccine sequence (KF487736)), and
four viruses DEV-vac, rDEV, rDEV-BAC and rDEV-Cre
DEV-vaccine strain in ducks and had no effect on its ability
to generate immunoprotection to lethal DEV challenge.
A process involving procedures “from cells to bacteria
and again back to cells” is experienced in constructing
the infectious DEV clone with the BAC system that could
potentially result in some changes to the viral genome. To
select a clone and/or virus that was closest to the parental
strain in terms of sequence, a series of studies, including
restriction enzyme digestion and genome sequencing
of the viral BAC clones, determination of rescued virus
plaque sizes and growth kinetics in vitro, and tests of
immunoprotection capability in ducks, were conducted.
Although there were a few mismatches and changes in
the viral genome in pDEV-vac compared with the
reference sequence, the obtained reconstructed
rDEVCre had almost identical immunoprotection
characteristics to the parental virus. Whether these mismatches
and changed sites have an effect on the virus in other
areas of characterization remains to be explored.
Two routine methods have been adopted to construct
recombinant virus containing a BAC sequence. One is to
co-transfect the transfer vector and viral genome into
cells (co-transfect method). The other is to transfect the
transfer vector into cells first and to then follow with a
virus infection (transfection-infection method). For cells
with low transfection efficiency, the latter is more efficient.
Primary chicken embryo fibroblasts are difficult to
transfect, and low transfection efficiency limits homologous
Figure 3 Multi-step growth curves of rDEV, rDEV-BAC,
rDEV-Cre, and DEV-vac. Comparison of the in vitro growth of
viruses reconstructed with parental DEV. The virus titers of
infectedcells (A) and supernatants (B) were determined at different times (0,
12, 24, 36, 48, 60, and 72 h) after inoculation of approximately 0.02
MOI of cell-free viruses of rDEV, rDEV-BAC, rDEV-Cre, and DEV-vac.
The multi-step growth curves were computed from three independent
recombination of duck enteritis virus genome to transfer
plasmids. In this study, we adapted the transfection of
suspended CEFs using the calcium phosphate method
to improve transfection efficiency.
In animal experiments, viral DNA was detected using
PCR of serum samples of all ducks immunized for
2 weeks without challenge with highly virulent DEV.
The results indicated that viral DNA still existed in the
blood of ducks in the immunized groups (data not
shown). This finding demonstrates that those viruses
were still active in ducks after being injected into the
body for 2 weeks, and that viruses lies in body for a
long time may be the prerequisite for live attenuated
vaccines to express viral antigens that periodically
Figure 4 Plaque area measurement of DEV-vac, rDEV, rDEV-BAC,
and rDEV-Cre on CEFs. The means and standard deviations of sizes
of 100 plaques of each virus were measured with Image J software.
The mean of the plaque area of DEV-vac was set at 100%. Standard
deviations are shown with the error bars.
stimulate the immune system to establish long-term
We established an infectious bacterial artificial
chromosome clone of a widely used attenuated live vaccine strain
in China. The results revealed that the rescued virus
has similar replication and immunogenic characteristics
as its parental strain. Based on this infectious clone, it
would be possible to develop novel vaccines, including
gene-deletion vaccines, or other marker vaccines for
serological differentiation of DEV naturally infected
from vaccinated animals. In addition, it would also be
possible to use a viral vector to construct a bivalent or
Figure 5 Protective efficacy of the reconstructed viruses
against lethal DEV challenge. Groups of 7 ducks were vaccinated
intramuscularly with 1 × 105 TCID50 of DEV-vac, rDEV, rDEV-BAC,
rDEV-Cre, or with culture medium as a control, and 2 weeks later, all
groups were challenged with lethal DEV. The ducks were monitored
daily for 2 weeks after challenge.
Materials and methods
All research was approved by the relevant committees at
the Zhejiang Academy of Agriculture Sciences.
Virus strain and cells
The DEV vaccine virus (C-KCE strain, a commercial DEV
vaccine in China, CVCC AV1221) and the standard highly
virulent CHv strain (CVCC AV1221) used in this study
were provided by the China Institute of Veterinary Drugs
Control (Beijing, China) . Chicken embryo fibroblasts
(CEFs) were prepared from 10-day-old
specific-pathogenfree (SPF) embryonated eggs (Zhejiang JianLiang
Bioengineering Co., Ltd., Hangzhou, China) according to
standard procedures and cultured in DMEM (Gibco-BRL)
supplemented with 8% FBS, 100 U of penicillin/mL and
100 μg streptomycin/mL.
Construction of transfer vector for homologous
Two pairs of primers, 18 F/18R and 15 F/15R (Table 3),
were designed to amplify the UL18 and UL15B fragments,
respectively, of the DEV-vac strain. The PCR products
were 1083 nt and 1258 nt in size, respectively. The
fragments were cloned into a pMD18-T vector (Takara)
using enzyme sites present in the primers, and the
resulting plasmid was designated pMD-UL15-UL18.
Then, a mini-F vector pHA2 containing xgpt and gfp
genes was released as a Pac I fragment from plasmid
pDS-pHAII, which was a gift from Dr. M. Messerle
, and cloned into the Pac I site present in
pMDUL15-UL18 to construct the transfer vector
pHA2UL18-UL15 (Figure 6).
Construction of the BAC clone of DEV-vac
Construction of the BAC clone of the DEV-vac strain
was conducted by inserting the bacterial mini-F plasmid
sequences flanked by loxP sites into the intergenic
region between genes UL15B and UL18 of DEV through
homologous recombination (Figure 6).
(1) Transfection of DNA into suspended CEF cells and
Table 3 Primers used in this study
Note. Restriction enzyme recognition sequences are underlined and italicized.
The transfer vector pHA2-UL18-UL15 was prepared
according to the protocol of the QiagenW plasmid
purification handbook (Qiagen) and then digested with Hind
III and precipitated with 1/10 volume of 3 M sodium
acetate and the DNA was dissolved in TE buffer at a
concentration of 0.5 μg/μL. A calcium phosphate method
was employed to transfect suspended cells according to
Morgan et al.  with modifications. In detail, primary
chicken embryo fibroblasts (CEFs) from 10-day-old SPF
chicken embryos were cultured in flasks for 16 to 24 h.
Once the cells had formed a confluent monolayer, the cells
were digested by trypsin-EDTA and seeded in a 6-well
plates with a density of 2.4 × 106 cells/well in growth
medium (DMEM containing 8% fetal calf serum)
without antibiotics. Transfection was operated according to
ProFection Mammalian Transfection System - Calsium
Phosphate (Promega, USA) protocol. 2 μg of linearized
transfer plasmid was transfected into suspended CEFs
per well. Following a 4-h incubation at 37°C, the cultures
were treated for 3 minutes with 15% glycerol in 1 × HBSP
(1.5 mL/well). Subsequently, a series of doses of DEV-vac
virus stock (200 μL, 100 μL, 50 μL, 25 μL,.) were added
to each well, and after 2 h of incubation at 37°C, the
culture medium was replaced with growth medium
(2) Selection of DEV recombinants expressing the xgpt
The DEV-vac-infected cells were collected when 80%
of the cells displayed CPE. Then, the collected cells were
added to one well of the 6-well-plate cells previously
incubated with selection medium containing 300 μg/mL
MPA (mycophenolic acid) (Sigma), 60 μg/mL xanthine,
100 μg/mL hypoxanthine, 2% FBS, 100 U of penicillin/
mL, and 100 μg streptomycin/mL The selection medium
was replaced at 1-d intervals. GFP-positive plaques were
isolated and transferred to newly prepared CEFs.
Following 10 cycles of plaque picking, the purified
recombinant virus rDEV with Xgpt and GFP labels was
(3) Generation and validation of a supercoiled
Figure 6 Schematic drawing of the cloning procedure to introduce the pHA2 vector into the DEV-vac genome. (A) Diagram of the
158 kb DEV-vac genome. Abbreviations: UL, unique long region; US, unique short region; TR, terminal repeat; IR, internal repeat. (B) The region of
interest from UL19 to UL15A genes in DEV-vac genome. (C) The HA2 segment was inserted between UL15B and UL18 by transfection of transfer
plasmid following infection with the DEV-vac genome. (D) The locations of the genes in pHA2 (including mini-F plasmid) are depicted by boxes.
xgpt, xanthine-guanine phosphoribosyltransferase; cat, chloramphenicol resistance gene; gfp, green fluorescent protein gene; rep E, replication
gene E; sopA, sopB, sopC, partitioning genes A, B, C; Pcmv, cytomegalovirus promoter. B, BamH I; P, Pac I; H, Hind III; E, EcoR I.
To generate the circular, supercoiled form of the BAC
clone, the cells infected with the recombinant virus in
the early phase (approximately 30-50% CPE) were collected,
and the total DNA was extracted from the cells using
the SDS-proteinase K method as described previously
. Then, 1 μg of DNA was electroporated into E. coli
DH10B competent cells (Invitrogen) according to the
manufacturer’s instructions. The transformed bacteria
were incubated in 1 mL of SOC medium for 1 h and
then plated on LB agar containing 30 μg/mL
chloramphenicol. The next day, single colonies were picked and
cultured in liquid LB medium, and BAC DNAs were
isolated by alkaline lysis of E. coli. Then, BAC DNAs of
pDEV-vac were identified by RFLPs, and the infectivity
of the pDEV-vac was examined by transfecting the
BAC DNA into CEF cultures.
Reconstitution and characterization of BAC-derived DEV
Next, 4 μg of pDEV-vac DNA was transfected into CEFs
by calcium phosphate precipitation as mentioned above.
The cells were then cultured with DMEM supplemented
with 8% FBS for 3–6 days. The virus collected was
named rDEV-BAC. The mini-F sequence in the
rDEVBAC genome was excised by co-transfecting 1 μg each
of the Cre recombinase expression plasmid
pCAGGSNLS/Cre (kindly provided by Dr. N. Osterrieder, Freie
Universität Berlin, Berlin, Germany) and pDEV-vac into
the CEF cultures. The virus rDEV-Cre without mini-F
vector sequences was obtained from the transfected CEF
cultures by limiting dilution and plaque purification and
then identified by PCR amplification with the primer pairs
18 F/15R (Table 3).
Multi-step growth kinetics of those reconstructed
viruses were determined and compared with the parental
virus DEV-vac. Briefly, the CEFs were infected with
approximately 0.02 MOI of cell-free viruses of rDEV,
rDEV-BAC, rDEV-Cre, and DEV-vac. The cells and
culture supernatant were harvested at different times
(0, 12, 24, 36, 48, 60, and 72 h) after virus infection.
The cells were collected by trypsin digestion following
two washes with phosphate-buffered saline (PBS, pH 7.0)
and were then suspended with 1 mL of DMEM-2% FBS
and an equal volume of supernatant. The collected cells
and supernatants were stored at −70°C until all samples
had been collected. Before titration on CEFs, the cells
were treated with a Tissuelyser-24 at 65 Hz for 60 s and
centrifuged at 4000 rpm for 5 min, and then 100 μL of
lysis supernatant and culture supernatant were taken
for titering via the TCID50 test according to standard
virological methods. The multi-step growth curves were
computed from three independent experiments.
The plaque sizes of rDEV, rDEV-BAC, rDEV-Cre,
and DEV-vac were also measured. The viruses were
serially diluted and plated onto CEFs seeded in a
12-well plate, and 2 h later, the culture medium was
replaced with DMEM containing 1.5% methylcellulose.
After a 4-day-incubation at 37°C, the cells were
treated with 5% formaldehyde solution and stained
with 1% crystal violet/70% ethanol. Then, for every
virus, 100 plaques were randomly selected and their
size measured using Image J software (http://rsb.info.
nih.gov/ij/). Statistical analyses to compare the
differences in the plaque sizes between the four strains
were conducted using one-way ANOVA with SPSS
Sequencing the BAC with Ion torrent technology
The sequence of pDEV-vac was determined with Ion
Torrent technology from the Invitrogen company. Briefly,
a DNA fragment library was first prepared with the
Ion Plus Fragment Library Kit (Life Technologies). The
resulting DNA library was then bound to ionic sphere
particles (ISPs) by specialized adaptors ligated onto the
ends of the fragmented DNA. Then, the DNA templates
on ISPs were amplified using PCR with primers specific
for the adaptors. Finally, sequencing was performed
using an Ion Torrent Personal Genome Machine (PGM)
(Life Technologies) platform, and the data were assembled
using Torrent Suite software (Life Technologies). The
obtained sequence was aligned with several published
whole genome sequences of DEV vaccine strains
[GenBank: EU082088.2, GenBank: KF487736, and GenBank:
NC_013036.1] with Vector NT1 Advance 9 software
(Invitrogen). The regions of the sequenced pDEV-vac
that were different from several reference nucleotide
sequences were amplified by PCR and sequenced again
using standard chain termination protocols. The ultimate
sites of difference between pDEV-vac and the reference
strain KF487736 were determined, and the corresponding
sites in four viral (DEV-vac, rDEV, rDEV-BAC, and
rDEV-Cre) genomes were also determined by PCR and
Vaccine efficacy in ducks
A total of 35 20-day-old specific-pathogen-free (SPF)
ducks (Harbin Veterinary Research Institute, CAAS,
China) were used for these studies. The animals were
randomly assigned to five groups (n = 7) and housed in
separate rooms. Four groups were separately inoculated
intramuscularly (i.m.) with 0.5 mL of medium containing
1 × 105 TCID50 (50% tissue culture infection dose) of
rDEV, rDEV-BAC, rDEV-Cre, and DEV-vac. The fifth
group served as a negative control and was inoculated
with medium without virus. Two weeks later, all groups
were challenged with a 100-fold 50% duck lethal dose
(DLD50) of a highly virulent DEV CHv strain i.m. Then, the
signs of disease and death were observed within 2 weeks.
DEV: Duck enteritis virus; CEFs: Chicken embryo embryoblasts; BAC: Bacterial
artificial chromosome; RFLPs: Restriction fragment length polymorphisms.
The authors declare that they have no competing interests.
LC planned and carried out virus recombinant, selection and identification of
recombinant BAC clone, and rescue of reconstructed viruses, prepared
figures, and drafted the manuscript. BY, JH and WY performed animal
experiment. TY performed characterization of viruses in cells. ZN carried out
viral detection and sequence alignment. XD constructed transfer vector for
homologous recombination. And CZ gave suggestion for this research and
helped with overall planning and drafting of the manuscript. All authors read
and approved the final manuscript.
The study was supported by grants from the International Science and
Technology Cooperation Project of Science Technology Department of
Zhejiang Province (2011C14011), Key scientific and technological innovation
team of Zhejiang Province (2010R50027) and the Special Fund for
Agroscientific Research in the Public Interest (201003012).
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