An inactivated gE-deleted pseudorabies vaccine provides complete clinical protection and reduces virus shedding against challenge by a Chinese pseudorabies variant
Wang et al. BMC Veterinary Research
An inactivated gE-deleted pseudorabies vaccine provides complete clinical protection and reduces virus shedding against challenge by a Chinese pseudorabies variant
Jichun Wang 0 1
Rongli Guo 0 1
Yongfeng Qiao 0 1
Mengwei Xu 0 1
Zhisheng Wang 0 1
Yamei Liu 0 1
Yiqi Gu 1 2
Chang Liu 1 2
Jibo Hou 0 1
0 Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses , Yangzhou , China
1 National Research Center of Engineering and Technology for Veterinary Biologicals/Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences , Nanjing, Jiangsu 210014 , China
2 College of Veterinary Medicine, Nanjing Agricultural University , Nanjing 210095 , China
Background: Since the end of 2011 an outbreak of pseudorabies affected Chinese pig herds that had been vaccinated with the commercial vaccine made of Bartha K61 strain. It is now clear that the outbreak was caused by an emergent PRV variant. Even though vaccines made of PRV Bartha K61 strain can confer certain cross protection against PRV variants based on experimental data, less than optimal clinical protection and virus shedding reduction were observed, making the control or eradication of this disease difficult. Results: An infectious clone of PRV AH02LA strain was constructed to generate a gE deletion mutant PRV(LA-AB) strain. PRV(LA-AB) strain can reach a titer of 108.43 TCID50 /mL (50% tissue culture infectious dose) on BHK-21 cells. To evaluate the efficiency of the inactivated vaccine made of PRV(LA-AB) strain, thirty 3-week-old PRV-negative piglets were divided randomly into six groups for vaccination and challenge test. All five piglets in the challenge control showed typical clinical symptoms of pseudorabies post challenge. Sneezing and nasal discharge were observed in four and three piglets in groups C(vaccinated with inactivated PRV Bartha K61 strain vaccine) and D(vaccinated with live PRV Bartha K61 strain vaccine) respectively. In contrast, piglets in both groups A(vaccinated with inactivated PRV LA-AB strain vaccine) and B(vaccinated with inactivated PRV LA-AB strain vaccine with adjuvant) presented mild or no clinical symptoms. Moreover, viral titers detected via nasal swabs were approximately 100 times lower in group B than in the challenge control, and the duration of virus shedding (3-4 days) was shorter than in either the challenge control (5-10 days) or groups C and D (5-6 days). Conclusions: The infectious clone constructed in this study harbors the whole genome of the PRV variant AH02LA strain. The gE deletion mutant PRV(LA-AB)strain generated from PRV AH02LA strain can reach a high titer on BHK-21 cells. An inactivated vaccine of PRV LA-AB provides clinical protection and significantly reduces virus shedding post challenge, especially if accompanied by the adjuvant CVC1302. While Inactivated or live vaccines made of PRV Barth K61 strain can provide only partial protection in this test.
Pseudorabies virus emerging variant; gE deletion; Inactivated vaccine; Adjuvant; Bacterial artificial chromosome; Challenge protection
Pseudorabies, caused by pseudorabies virus(PRV), has
leaded to significant economic losses to the pig industry in
many countries. Pseudorabies is a porcine infectious
disease that can be effectively controlled using gene deletion
marked vaccines and serum monitoring, enabling
differentiation of vaccinated from infected animals [1–3]. However,
in 2011 an outbreak of pseudorabies affected Chinese pig
herds that had been vaccinated with the standard PRV
Bartha K61 strain [4–9]. It is now clear that the outbreak
was caused by an emergent PRV variant [5–7, 9–13].
As a support for this finding, the widely used PRV
Bartha K61 strain was shown to be incapable of
providing complete protection against this new PRV
variant in several experimental studies [4–8]. Thus, a more
efficacious vaccine is perceived as a necessary tool to help
affected herds revert to PRV virus negative status.
To develop an effective vaccine, gene deletion mutants
were generated from the new PRV strain and evaluated
for protection efficacy. Although live vaccine candidates
based on gE, gE/gI, or TK/gE/gI gene deletion mutants
are assumed to be highly protective, the safety issues of
these preparations have to be demonstrated by lengthy
testing [4, 14–18]. Alternatively, inactivated version of
vaccine from the same mutants are promising to provide
clinical protection as well based on relevant data about
inactivated Bartha strain.
In this study, LA-AB, a gE deleted mutant of the
emerging PRV strain AH02LA, was constructed using a
bacterial artificial chromosome clone. A vaccine utilizing
this mutant was evaluated with and without adjuvant in
regard to its ability to provide clinical protection and
reduce virus shedding after lethal challenge.
Cells and viruses
BHK-21 cells (CVCC:CL5, from China Veterinary Culture
Collection Center) and chicken embryo cells (CECs) (SPF
chicken embryo eggs were from Beijing Merial Vital
Laboratory Animal Technology Co., Ltd) were
prepared using standard methods. The PRV Bartha K61
strain was kindly provided by Professor Ping Jiang of
Nanjing Agricultural University. Virus cultures and
stocks were prepared using CECs or BHK-21 cells before
at -70 °C. Virus titers were determined by TCID50 on
BHK-21 cells following a standard protocol. Viral DNA
extraction and transfection of plasmid, virus or bacterial
artificial chromosome(BAC) DNAs were performed as
described previously [19, 20].
Plasmid and BAC manipulation
The mini-F transfer vector pHA2-pUC19-H1-H2 was
constructed as described . Plasmid and BAC DNAs
were isolated using commercial kits (Invitrogen PureLink®
Quick Plasmid Miniprep and Qiagen Large-Construct Kit)
following the manufacturer’s instructions. Next, restriction
fragment length polymorphism(RFLP) analyses of PRV
BACs was conducted using the restriction endonucleases
BamH I, Kpn I, Pst I and Sph I and digestion conditions
recommended by the supplier (Takara). Electrocompetent
E.coli cells (DH10B) were obtained commercially
(Invitrogen). E.coli GS1783 was made electrocompetent using
published protocols [21–23]. Electroporation was
performed to transform viral or BAC DNA following
established methods [23–25].
PCR and sequencing
A pair of primers (Table 1) was designed from reference
sequence (GenBank:NC_006151.1) to amplify sequences
Table 1 Primers for PCR or sequencing
of gI gene with isolated PRV AH02LA strain DNA as
template. These primers were also used for sequencing gI
gene (GeneScript, Nanjing China). To obtain the gE
deletion, overlap PCR was conducted with two pairs of
primers, ΔgE TV overlap1 and ΔgE TV overlap2 (Table 1).
Briefly, with PRV AH02LA DNA as template, two PCR
reactions were performed separately with the above two
primer pairs. Next, the PCR products were gel purified to
serve as templates in another PCR reaction with primers
ΔgE TV overlap1 F and ΔgE TV overlap2 R to produce a
fragment for gE deletion mutant generation. Thus the
generated mutant, consists of upstream sequences, the
intact gI gene, part of gE gene(1299 bp to 1735 bp of gE
ORF), and downstream sequences (Fig. 1). Finally, the
primers PRV Homo-1 F and PRV Homo-2R  were
used to amplify the repairing viral DNA with the PRV
AH02LA DNA as template.
Multistep growth kinetics
Multistep growth kinetics of parental AH02LA, gE
deletion LA-AB, and repair AH02LAR viruses were tested on
BHK-21 cells following methods as described  on
monolayers (1 × 106 cells) of BHK-21 cells with a
multiplicity of infection(MOI)of 0.01. Three independent
experiments were conducted and one-way ANOVA (SPSS
LD50 determination for PRV AH02LA strain
To determine the LD50 of the virulent PRV AH02LA
strain, the virus (TCID50 = 10-8.25/mL) was serially
diluted with DMEM to 10-1, 10-2, 10-3 and 10-4.
Twentyfive 77-day-old piglets were randomly divided into five
groups. Piglets in each challenge group were inoculated
intranasally(I.N.) with 1 mL diluted virus of different
titers. Animals were observed for additional fourteen days.
The number of piglets that died in each group was
recorded to calculate LD50 using the Karber method.
Construction of PRV BAC and deletion mutants
The PRV BAC was constructed from an isolate of the
emergent PRV variant following published methods (Fig. 1)
[16, 26]. Briefly, After cotransfection of ~1.5 μg PRV
AH02LA strain DNA and ~4 μg pHA2-pUC19-H1-H2
DNA, mini-F recombinant viruses with green fluorescence
were purified to obtain a homogeneous population .
Next, mini-F containing PRV DNA was isolated and then
transferred into E. coli cells DH10B and subsequently into
E.coli strain GS1783 cells by electroporation for
mutagenesis . Finally, BAC DNA was isolated and RFLPs were
identified as described  using BamH I, Kpn I, Pst I and
Fig. 1 Construction of mini-F recombinant PRV AH02LA strain and its gE deletion mutant virus (LA-AB). a The whole genome of PRV. b Homologous
recombination was conducted to insert mini-F in lieu of gI and gE to generate the mini-F recombinant PRV AH02LA strain for BAC construction.
c Another recombination was performed to recover the whole gI gene and part of gE gene, the resulting BACPRV-G was used to generate the gE
deletion LA-AB strain. Scales in bp or kbp are provided
Sph I, using the complete genome sequence of PRV ZJ01
strain (GenBank: KM061380.1) as a reference for in silico
To generate the gE deletion mutant of PRV AH02LA
strain, another homologous recombination was
performed to recover the gI gene and the undeleted
sequences of gE as shown in Fig. 1. Briefly, PCR with
primers ΔgE TV overlap1 F and ΔgE TV overlap2 R
(Table 1) was performed with the PRV AH02LA DNA as
template. And then CECs were co-transfected with
approximately 5 μg PRV BAC DNA and 2 μg DNA of
fragment from the second PCR reaction of overlap PCR
with primersΔgE TV overlap1 F andΔgE TV overlap2 R
by calcium phosphate precipitation. Using illumination
at 488 nm, non-fluorescent virus isolates were selected
and purified to homogeneity. Product structure was
confirmed by sequencing with the same primers and other
specific primers from the SEQ-gI/gE series (Table 1).
For generation of the gI/gE repair virus via
homologous recombination, CECs were transfected with
approximately 5 μg PRV BAC DNA and 2 μg PCR product
generated using DNA of parental PRV as template and
primers matching PRV Homo 1 F and PRV Homo 2R
 (Fig. 1). Two to three days after transfection,
nonfluorescent plaques (488 nm) were isolated and purified
to obtain a homogeneous population. The structure of
the recovered viruses was confirmed by PCR and
sequencing with primers PRV Homo 1 F, PRV Homo 2R
and sequencing primers of SEQ-gI/gE (Table 1).
Preparation of vaccine
PRV LA-AB and Bartha K61 viruses were propagated in
BHK-21 cells using a 5 liter bioreactor, each virus stock
was diluted to 108.5 TCID50/mL. After inactivation with
formalin (Sigma-Aldrich) as described , vaccines were
prepared using mineral oil adjuvant (1:3 water in oil). All
vaccines were confirmed to be free of bacteria and fungi,
and had the expected physical properties according to
standard protocols. Vaccines were stored at 4 °C until use.
Test of vaccine efficacy
Thirty piglets of 21-day-old (from Zhengzhuquan Pig
Breeding Farm in Pukou district, Nanjing, China) were
randomly divided into 6 groups (A-F) and housed in
separated facilities. All piglets were antibody negative for
PRV gB and gE, and free of porcine reproductive and
respiratory syndrome virus, porcine parvovirus, porcine
circovirus 2 and classical swine fever virus. All
inoculations were given intramuscularly(I.M.) at 2 mL/pig.
Group A piglets were inoculated with LA-AB, group B
piglets with LA-AB plus adjuvant CVC1302 (Patent:
CN103083663B, kindly provided by the group of Dr.
Qisheng Zheng in our institute), group C piglets with
inactivated Bartha K61, group D piglets with live Bartha
K61 of 1 × 105.0 TCID50 virus/dose, and groups E and F
piglets were dosed with PBS only. Piglets in groups A, B,
and C were inoculated at 28 days of age and again at
56 days. Group D piglets were inoculated once, at
28-dayold. Group E and F piglets were dosed once, at 28 days of
age. Three weeks after the second inoculation (at which
time the piglets were 11-week-old), groups A, B, C, D and
E were challenged I.N. with 3 × LD50 PRV AH02LA per
piglet. Group F piglets were not challenged, serving as a
negative control. Serum samples from all piglets were
collected before inoculation, and at 7, 14, 21, 28, 35, 42 and
49 days post inoculation, and again at 14 days post
challenge(P.C.). Serum was subjected to ELISA tests for PRV gB
and gE antibodies, and cross-neutralizing (NA) antibodies
against AH02LA or Bartha K61. Body temperature, clinical
signs, and virus shedding were monitored and recorded
daily until 14 days P.C.. The presence of lung lesions for
died or survived piglets was noted at the end of the test.
Test for serological antibodies
ELISA tests were performed with the PRV gE or gB
antibody detection kit (IDEXX, Maine, USA) following
manufacturer’s instructions. Cross neutralization tests were
conducted according to the standard methods published
by OIE (Veterinary Pharmacopoeia of the People's
Republic of China (2010)) with slight modification. Briefly,
serum samples were diluted two-fold with PBS, mixed
with 100 TCID50 PRV AH02LA or PRV Bartha K61
virus, and incubated for 1 h at 37 °C. The serum-virus
mixture was used to inoculate BHK-21 cells which were
then incubated at 37 °C, 5% CO2 for 3–4 days to observe
the development of CPE. Titers of neutralizing antibody
were expressed as nlog2 of highest dilution at which no
CPE was observed, and mean titers of each group were
calculated for comparison.
Detection of virus shedding
Nasal swab samples were collected from all piglets
before challenge and daily to 14 days after challenge. After
shaking at 5000–8000 RPM for 1–2 min, one
freezethaw cycle (-70 °C and 37 °C) was conducted to release
viruses from nasal swabs. Samples were centrifuged at
10,000 RPM for 15 min to pellet tissue and cell debris,
the supernatants were collected to determine virus titers.
Viruses titers were expressed as TCID50 following the
method of Karber.
All animal studies were conducted following guidelines
provided by the Institutional Biosafety Committee and
approved by the Institutional Animal Care and Use
Committee at the Jiangsu Academy of Agriculture
Sciences. Experiments involving virulent PRV were
conducted under Biosafety Level 2+ containment.
Isolation and identification of an emergent pseudorabies
virus variant (AH02LA strain)
BHK-21 cells, inoculated with a sample of brain tissue
from a stillborn piglet, exhibited cytopathogenic effects
(CPE) after 3 days. After three rounds of plaque
purification, a homogeneous population of virus was isolated
and designated as the AH02LA strain. Using the viral
DNA as template and the primers PRV gI F/ R (Table 1),
PCR generated a product of 1250–1400 bp. Sequencing
established that this fragment was homologous to the
PRV gI gene. The AH02LA strain can be specifically
neutralized with the standard antibodies against PRV
(CIVDC, Beijing China) with a neutralizing index of
10,000. These results demonstrate that a variant PRV
field isolate was identified.
Pathology of the PRV AH02LA strain
The LD50 of PRV AH02LA (Batch: JJ-R5) was
determined using 77-day-old piglets free of antibodies against
PRV. Three of five piglets in the 10-5 dosage group, and
all of the piglets in the 10-4 dosage group, showed typical
symptoms of PRV infection: sneezing, purulent nasal
discharge, difficulty in breathing and ataxia, indicating
the high virulence of this strain. The LD50 was 10-2.32/
mL. The highly virulent character of AH02LA is shared
by other emergent PRV strains isolated during the 2011
outbreaks [4–8]. The significant difference between
these emerging strains and the conventional strain S
(HWBD, Harbin China) is that the former cause
mortalities in not only young piglets but also much older pigs,
whereas the conventional strain S causes death only in
young (less than 15-day-old) piglets (Veterinary
Pharmacopoeia of the People's Republic of China
Construction of a bacterial artificial chromosome
containing the AH02LA genome
Green plaques of mini-F recombinant PRV AH02LA
were observed under UV light (488 nm), and isolated
after several rounds of plaque purification. The mini-F
recombinant PRV AH02LA DNA was isolated and
transfected into Escherichia coli DH10B competent cells, the
resulting clone was designated BACPRV-AH02LA.
BACPRVAH02LA DNA was electroporated into E. coli GS1783
competent cells, resulting in a clone designated BACPRV-G.
This BAC was used for further gene manipulations using
the En Passant protocol . Restriction fragment length
polymorphism (RFLP) analysis of BACPRV-G with BamH I,
Kpn I, Pst I and Sph I generated patterns similar to those
predicted from the PRV ZJ01 strain sequence (GenBank:
KM061380.1) with minor differences (Fig. 2). Virus was
rescued successfully by transfection of the BACPRV-G
DNA into primary CECs, designated PRV AH02LAR. In
summary, an infectious clone containing the complete
genome of the PRV emerging variant AH02LA strain was
Fig. 2 Plaques of mini-F recombinant PRV AH02LA strain and RFLP of BACPRV-G. a Images of mini-F recombinant PRV AH02LA and the parental
AH02LA plaques under UV excitation (upper) and phase contrast (lower) are shown. Arrowhead shows a plaque of parental PRV AH02LA virus
and arrow shows a plaque formed by mini-F recombinant PRV AH02LA. Each panel represents a view of 200 × 200 μm in size. b RFLP of BACPRV-G,
DNA from PRV AH02LA BAC clone BACPRV-G was prepared by mini-prep and digested with BamH I, Kpn I, Pst I and Sph I (lanes 1–4). The digests
were separated by 0.8% agarose gel electrophoresis for 16 h under 40 V (Left). Predicted RFLP patterns of BACPRV-G with BamH I, Kpn I, Pst I and
Sph I digestion respectively. Predictions of these digestions were performed with the whole genome sequence of PRV ZJ01 strain (GenBank:
KM061380.1) as reference. M: DL 15,000 DNA Marker (Takara)
Construction of a gE deletion mutant virus
After co-transfection of BACPRV-G DNA with a DNA
fragment containing the gE deletion (obtained through
PCR), several white plaques were observed under UV
illumination at 488 nm. One was isolated after several
rounds of plaque purification and designated PRV
LAAB. The deletion was verified by PCR using primers of
ΔgE TV overlap1 F andΔgE TV overlap2 R, followed by
sequencing with the appropriate primers (Table 1).
Growth kinetics of the PRV LA-AB virus
Multistep growth kinetics for the PRV LA-AB, AH02LA,
and AH02LAR viruses were determined. Peak
supernatant titers for LA-AB, AH02LA, and AH02LAR were
108.18, 109.01, and 108.19 TCID50 /mL respectively. At 48
hpi the titers for LA-AB and AH02LAR were not
significantly different from AH02LA (p = 0.414 and 0.362). For
cell-associated virus titers LA-AB, AH02LA, and
AH02LAR were 108.43, 108.86, and 108.13 TCID50 /mL at
48 hpi, respectively. The titers of LA-AB and AH02LAR
are not significant different from the parental virus
AH02LA at 48 hpi (p = 0.126 and 0.088).
Immunogenicity of the LA-AB vaccine
In challenge control group, all piglets had typical clinical
syndromes of pseudorabies infection including sneezing,
coughing, difficulty breathing and nasal discharge P.C..
Two piglets died at 7 days and two at 8 days P.C. (Table 2).
Body temperatures of these piglets reached over 41 °C and
lasted for 4–5 days (Fig. 3(a)). All the piglets shed virus
with titers of 100.25 ~ 105.25 TCID50 /0.1 mL lasting for 5–
7 days till death or 10 days for the surviving piglet
(Fig. 3(b)). All piglets showed severe lung lesions from
hemorrhaging and congestion (Table 2).
gB antibodies were detected in all piglets vaccinated
with live Bartha K61 strain or the LA-AB strain plus
adjuvant at 7 days post vaccination(P.V.), while two of five
piglets vaccinated LA-AB strain alone or the inactivated
Bartha K61 strain were anti-gB positive at 7 days P.V..
All samples collected at 14 days P.V. were positive for
gB antibodies (Table 2). This result indicates the
adjuvant CVC1302 helps the LA-AB vaccine stimulate
immunity against PRV more quickly. At 14 days P.C. all
piglets were positive for gE antibodies (Table 2).
No neutralizing antibodies (NA) against the AH02LA or
Bartha K61 strain, were detected in any of the piglets at
7 days P.V.. At 14 days P.V. neutralizing antibodies were
detected in one piglet from group B (LA-AB plus
adjuvant) at a titer of 2. In groups A, B, and C, NA titers began
increasing quickly seven days after the boost and
continued to increase over the next two weeks. The LA-AB
vaccines induced higher titers of NA against the AH02LA
and Bartha K61 strain, than vaccines made of Bartha K61.
One dose of live Bartha K61 vaccine stimulated a weak
NA titer of less than 4 against the AH02LA strain or the
Bartha K61 strain. These results indicate that optimizing
the prime-boost regimen is necessary for live Bartha K61
vaccine to stimulate strong humoral immunity. NA titers
in group B piglets were higher than other groups,
indicating that adjuvant CVC1302 helped stimulate higher
specific immunity for LA-AB vaccine (Table 2).
All piglets in the vaccinated groups (A, B, C and D)
were protected against lethal challenge. Fevers in group
A and B piglets were transient, and other clinical
symptoms were mild or absent respectively. Titers of shed
virus in LA-AB vaccinated group A piglets were 100.25 ~
103.75 TCID50/0.1 mL lasting 3–4 days; in LA-AB plus
adjuvant group B piglets, titers were 100.125 ~ 102.875
TCID50/0.1 mL lasting 3–4 days. Piglets in groups C and
D (vaccinated with inactivated Bartha K61 and live
Bartha K61 respectively) presented fevers of more than
41 °C lasting for 4–6 days (Fig. 3(a)), four and three
piglets respectively, presented sneezing lasting for 2–4 days
and nasal discharge lasting 2–3 days (Table 2). The titers
of shed virus in these piglets were 100.125 ~ 104.125
TCID50 /0.1 mL lasting for 5–6 days (Fig. 3(b)). No Lung
lesions were observed in any group A or B piglets, while
2/5 group C piglets and in 1/5 group D piglets presented
lesions from hemorrhage and congestion (Table 2).
The results of vaccination test demonstrate that vaccines
made of PRV LA-AB elicit a more effective immune
response against the emergent PRV AH02LA than the
Bartha K61 vaccines. The immune response is even more
pronounced when PRV LA-AB is administered with
adjuvant. Pigs vaccinated with LA-AB produced lower titers of
virus shedding over a shorter duration than pigs with
Bartha K61. This is a clear advantage in the terms of
controlling the spread of infection. Adjuvant CVC1302 is a
mixture of monophosphoryl lipid A (MPLA), muramyl
dipeptide (MDP) and β-glucan. This mixture has been
shown to improve the efficiency of the inactivated
vaccines of porcine epidemic diarrhea virus and foot and
mouth disease virus (Patent:CN103083663B). MPLA,
MDP and β-glucan can serve as delivery systems or
stimulators for the immune system [28–31].
Bacterial artificial chromosome(BAC) of herpesvirus is
a useful tool for generation of gene modified mutants to
study the mechanism of the viruses. After the
construction of the first BAC of mouse cytomegalovirus
(MCMV) , a few of herpesvirus genomes have been
maintained in BACs as infectious clones, which have
provided great help for discovery of pathology of these
viruses [27, 33] and construction of vectored vaccines
[26, 34, 35], especially following the protocol of En
Passant protocol . The parental PRV AH02LA strain of
the BAC constructed in this study is a virulent variant
strain that caused severe losses in many porcine farms in
China. So even though En Passant protocol was not
performed in the generation of the gE deleted LA-AB strain
in this study, it will be used for further analysis of the
mechanism of the high virulence of this variant.
The latent infection of PRV is always a potential threat
to porcine herds . So the eradication of PRV has been
a direct aim of many farms for control of this disease. This
aim has been achieved in a few countries  and the
infection of wild PRV has been controlled to very low levels in
some individual farms in China before 2011 [37, 38].
DIVA vaccine(vaccine allowing differentiating infected
from vaccinated animals) has played an important
role towards this aim . In the project of eradication
of PRV, the efficient reduction of virus shedding post
infection with virulent strain is crucial . In this
study, the inactivated vaccine of LA-AB strain with
adjuvant CVC1302 reduced virus shedding
significantly, despite that all vaccinated animals did not
stop virus shedding post challenge.
We did find a difference in the profile of clinical
symptoms between animals challenged through I.M. or I.N.
route. During the challenge test in this study, we also
tried the challenge route of I.M. injection for
11-weekold piglets (data not shown). We found that even though
it caused high fever and obvious clinical syndromes of
PR through this way, the virulence was significantly
Fig. 3 Body temperatures and virus shedding post challenge with PRV AH02LA strain. a Body temperatures of piglets in vaccinated groups were
detected together with challenge control and placebo control from one day before challenge to 14 days post challenge. Average temperatures
of five piglets of each group were taken for comparison. Error bars represent the standard deviations. b Nasal swab samples of all piglets of
vaccinated groups together with challenge control and placebo control groups were collected before challenge and daily up to 14 days after
challenge for virus isolation and titer determination. The titers of viruses were expressed as TCID50 on BHK-21 cells following the method of
Karber. Average titers were determined with the samples that virus exceeding were detected
lower than through I.N. way. This difference is
presumed to be associated with the increased titers of virus
after replication on epithelial cells of respiratory tract.
To mimick the nature infection of domestic pigs
genuinely, we did the challenge protection test through I.N.
way for evaluation of the vaccines in this study.
The infectious clone consists of the whole genome of PRV
emerging variant AH02LA strain. Inactivated vaccine
made of PRV LA-AB strain can provide better clinical
protection than vaccines made of Bartha K61. In addition,
significant decrease of virus shedding was observed post
lethal challenge when LA-AB inactivated vaccine were
adjuvanted with CVC1302.
BAC: Bacterial artificial chromosome; CEC: Chicken embryo cell;
CPE: Cytopathogenic effects; E.coli: Escherichia coli; ELISA: Enzyme-linked
immunosorbent assay; gB: glycoprotein B; gE: glycoprotein E; gI: glycoprotein
I; Hpi: Hour post infection; I.M: Intramuscularly; I.N: Intranasally; LD50: 50%
lethal dose; MCMV: Mouse cytomegalovirus; MDP: Muramyl dipeptide;
MOI: Multiplicity of infection; MPLA: Monophosphoryl lipid; NA: Neutralizing
antibodies; ORF: Open reading frame; PC: Post challenge; PV: Post vaccination;
PBS: Phosphate buffer saline; PCR: Polymerase chain reaction; PRV: Pseudorabies
virus; RFLP: Restriction fragment length polymorphism; TCID50: 50% tissue
culture infectious dose; TK: Thymidine kinase; VN: Virus neutralization
This study was supported by the Special Fund for Agro-Scientific Research in
the Public Interest (201303046), Program for Independence and Innovation
in Agricultural Sciences of Jiangsu Province (CX(12)3061 and CX(14)2084),
and the Natural Science Foundation of Jiangsu Province (BK20131334).
Availability of supporting data
The data set(s) supporting the results of this article is included within the
article. And all row data are available in National Research Center of
Engineering and Technology for Veterinary Biologicals/Institute of Veterinary
Medicine, Jiangsu Academy of Agricultural Sciences, Nanjing Jiangsu, China
upon official request via mail ().
JW and JH designed the generation of BAC and related mutants. JW, YQ, YG
and CL contributed to the design and performance of animal tests. RG, YG,
CL, YL and MX constructed the bacterial artificial chromosome of PRV and
the related studies. JW ZW and RG constructed the gE deleted vaccine. ZW,
JW, YQ and RG investigated the related properties of the vaccine and
performed the animal experiments and related tests. All authors approved
the final manuscript.
All animal tests were approved by the Institutional Animal Care and Use
Committee and were conducted following the guidelines of the Institutional
Biosafety Committee at the Jiangsu Academy of Agriculture Sciences.
Experiments involving virulent PRV were conducted under Biosafety Level 2+
containment. At the end of test, all survived piglets were euthanized by
injection of pentobarbital.
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