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Vaccine 31 (2013) 18381847
Contents lists available at SciVerse ScienceDirect
Vaccine
j ournal homepage: www.elsevier .com/ locate /vaccine
Synthetic B- and T-cell epitope peptides ofporcine reproductive and respiratorysyndrome virus with Gp96 as adjuvant induced humoral and cell-mediatedimmunity
Caiwei Chen a,b,Jing Li a, Yuhai Bi a, Limin Yang a, Shanshan Meng a, Yuancheng Zhou a,XiaojuanJia a, Songdong Meng a, Lei Sun a, Wenjun Liu a,b,
a Key Laboratory of Pathogenic Microbiologyand Immunology, Institute ofMicrobiology, Chinese Academy of Sciences,Beijing 100101, Chinab University of Chinese Academy of Sciences,Beijing 100101, China
a r t i c l e i n f o
Article history:
Received 21 January 2012
Received in revised form 6 January 2013
Accepted 25 January 2013
Available online 7 February 2013
Keywords:
HP-PRRSV
Synthetic peptide vaccine
B-cell epitope
T-cell epitope
Humoral and cell-mediated immunity
Gp96
a b s t r a c t
Highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) has recently caused
huge economic losses in the pig industry worldwide. Commercial vaccines, including inactivated vac-
cines and attenuated live vaccines, are available but fail to provide sustainable protection, especially
against genetically heterologous strains. Thus several approaches have been used to develop more effec-
tive PRRSV vaccines and/or immune modulators to accelerate and magnify immune responses to PRRSV
vaccines. Heat shock protein Gp96 is one such modulator that enhances both the innate and adaptive
immune responses. In the present study, two B-cell epitopes and seven T-cell epitopes from PRRSV and
a Pan DR T-helper cell epitope were synthesized and mixed with the N-terminal 22355 aa of Gp96
(Gp96N) as an adjuvant, and immune responses were evaluated. Our results show that Gp96N activated
PRRSV-specific humoral immune responses elicited by BCE-peptides and promoted the PRRSV-specific
cellular immunity induced by TCE-peptides. Moreover, higher levels ofIL-12 and TNF- and lower levels
ofIL-4 and IL-10 were observed in the serum ofGp96N-vaccinated piglets compared to piglets immunized
with no Gp96N, displaying a predominant Th1 type ofimmune response induced by Gp96N. Following
challenge with the virulent HP-PRRSV isolate JXwn06, piglets vaccinated with the mixture of peptidesand Gp96N presented with milder clinical symptoms, lower viremia, and less pathological lesions in their
lungs, however, this vaccine could not provide lasting and effective protection against HP-PRRSV infec-
tion. These data provide important bases for the development ofPRRSV epitope-based synthetic peptide
vaccines combined with Gp96N as attractive immunomodulators in swine.
2013 Elsevier Ltd. All rights reserved.
1. Introduction
Porcine reproductive and respiratory syndrome virus (PRRSV)
has two major genotypes: the European type (type I; the Lelystad
strain as the prototype) and the North American type (type II;
the ATCC VR2332 strain as the prototype), both of which belong
to the Arteriviridae family, order Nidoviridales [1]. PRRSV, espe-
cially highly pathogenic PRRSV (HP-PRRSV), causes more than
two million cases of pig infection annually in China [2] and costs
the US pork industry approximately 664 million dollars in direct
losses per year [3]. Several approaches to develop PRRSV vaccines
have been evaluated, including live attenuated virus, inactivated
Corresponding author at: Center for Molecular Virology, Institute of Microbiol-
ogy, Chinese Academy of Sciences, Beijing 100101, China. Tel.: +86 10 64807497;
fax: +86 10 64807503.
E-mail address: [email protected](W. Liu).
virus, recombinant subunit vaccine systems (based upon plasmid
DNA, bacteria, baculovirus, adenovirus, fowlpox virus, and pseu-
dorabies virus carrying several PRRSV structural proteins) and
chimeric viruses [49]. However, commercially available vaccines
have limited effect against heterologous virus challenges with
potential to revert to virulence [410].
It has been shown that synthetic peptides possess the capa-
bility to elicit critical epitope-specific immune responses without
adverse effects from the vaccines mentioned above. Advancements
in molecular biology, molecular immunology, and bioinformatics
have enhanced the discovery of antigenic epitopes of PRRSV. To
date, aa 3745 of the major envelope glycoprotein Gp5 is the main
target for viral neutralizing antibodies (NAs) in both the geno-
types of PRRSV [11]. In addition, type I PRRSVs strongly induce NAs
against aa 5768 of Gp4 [12,13], but whether this B-cell epitope
(BCE) from type II PRRSV strains can induce NA remains unclear.
Recently, two T-cell epitopes (TCEs) in Gp4 and three TCEs in the
nucleoprotein (N) of type I PRRSV strains, as well as two TCEs
0264-410X/$ seefrontmatter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.vaccine.2013.01.049
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C. Chen et al. / Vaccine31 (2013) 18381847 1839
Table 1
Characteristics of theB- and T-cell epitopes previously reported andused in thepresent study.
Epitope ID Reported information Synthetic peptide sequences in this study
aAA position and sequences Reference
Gp4-59B 59 SAAQEKISF67 (b I) [12,13] 59 SSPTIRKIS
Gp5-37B 37 SHL/FQLIYNL45 (cII) [11] 37 SHIQLIYNL
Gp4-7T 9FLLAGAQHI17 (II) [15] 7FLLVGFKCF
Gp4-170T 170 CLFAILLAT178 (II) 170 CLFAILLAI
Gp5-117T 117 LAALICFVIRLAKNC131 (II) [14,15] 117 LAALICFVIRLAKNC
Gp5-149T 149 KGRLYRWRSPVII/VEK163 (II) 149 KGRLYRWRSPVIVEK
N-49T 49 KPEKPHFPL57 (II) [15] 49 NPEKPHFPL
N-63T 63 VRHHLTQTE71 (II) 63 VRHHFTPSE
N-104T 104 FMLPVAHTVRLIRVTST120 (II) 104 FSLPTQHTVRLIRATASdPADRE AKFVAAWTLKAAA [27] AKFVAAWTLKAAA
The differentaa of theepitope used in the present study are marked by underlining.a AA, amino acid.b I, type I PRRSV.c II, type IIPRRSVd PADRE,Pan DR T-helper cell epitope.
in Gp5 of both types of strains were identified based on their
ability to induce IFN- secretion in cultures of peripheral blood
mononuclear cells (PBMCs) obtained from PRRSV-immunized pigs
[14,15]. Regardless, it is still unknown if these two BCE and seven
TCE peptides can induce PRRSV-specific and protective immuneresponses in pigs.
Due to the delayed and weak cell-mediated immunity and NA
production of the current PRRSV vaccines [1618], many immune
modulators have been used to provoke stronger immune responses
to experimental vaccines in pigs, including CD40L, GM-CSF, HSP70,
and CpG [68,19]. The heat shock protein (HSP) Gp96, as well as
its N-terminal fragments (aa22355,Gp96N), are able to bind pep-
tides and act as potent immuno-adjuvants to enhance both innate
and adaptiveimmune responses in mice models [2023]. However,
whether this fragment of porcine Gp96 can play the same role in
pigs remains to be determined.
In this study, bioinformatics were used to align the above BCEs
and TCEs among the type II PRRSVs, andconsensus sequences were
obtained. Humoral and cellular immune responses and the protec-tive efficacy of BCE + TCE-peptides with the Gp96N adjuvant were
investigated in piglets.
2. Materials and methods
2.1. Cell lines, viruses, and antibodies
MARC-145cells weremaintained in Dulbeccos modified Eagles
medium (DMEM, GIBCO) supplemented with 10% heat-inactivated
fetal bovine serum (FBS, GIBCO) and were used to propagate and
titer HP-PRRSV isolate JXwn06 (a kind gift of Prof. Hanchun Yang of
China Agricultural University, GenBank accession No. EF641008.1).
HRP-conjugated and TRITC-conjugated anti-swine IgG were pur-
chased from Santa Cruz.
2.2. Synthetic peptides and purification of Gp95N
According to the aa sequences of each BCE and TCE shown in
Table 1, Gp4-59B (SSPTIRKIS, purity: 98.4%), Gp5-37B (SHIQLIYNL,
purity: 98.6%), Gp4-7T (FLLVGFKCF, purity: 97.8%), Gp4-170T
(CLFAILLAI, purity: 97.4%), Gp5-117T (LAALICFVIRLAKNC, purity:
87.5%), Gp5-149T (KGRLYRWRSPVIVEK, purity: 85.9%), N-49T
(NPEKPHFPL, purity: 96.7%), N-63T (VRHHFTPSE, purity: 97.2%), N-
104T (FSLPTQHTVRLIRATAS, purity: 81.2%), and a Pan DR T-helpercell epitope (PADRE) peptide (AKFVAAWTLKAAA, purity: 82.0%)
were all synthesized and purified at Scilight Biotech, Beijing, China.
Gp4-59B and Gp5-37B were coupled to the carrier protein keyhole
limpet hemocyanin (KLH) or bovine serum albumin (BSA) by the
same company.
Recombinant Gp96N was expressed in Escherichiacoli and puri-
fied using nickel-nitrilotriacetic acid (Ni-NTA) chromatography
(GE). Endotoxins in the protein preparation were removed using
a bacterial Endotoxin Kit (Hycult Biotech).
2.3. Pig immunization and HP-PRRSV challenge
Sixteen 4-week-old piglets were obtained from the BeijingCenter for SPF Swine Breeding & Management. All piglets were
negative for anti-PRRSV antibodies as demonstrated by commer-
cial ELISA kit (Herdcheck, IDEXX). The animals were also negative
for porcine circovirus type 2 (PCV2), Classical Swine Fever Virus
(CSFV), Pseudorabies virus (PRV), and PRRSV by PCR and/or RT-
PCR. Then, piglets were randomly separated into four groups (four
piglets per group) and housed at Ceva-China Biotech, Beijing.
Piglets were intramuscularly injected three times at 2-week inter-
vals according to Table 2. Sera and PBMCs separated from the
blood at 0, 14, 28, and 42 days post injection (dpi) were ana-
lyzed by ELISAs, lymphocyte proliferation assays, and cytokine
assays.
Then, the vaccinated piglets were intranasally challenged with
2105 TCID50
HP-PRRSV JXwn06 at 42dpi and monitored daily
until they died. Rectal temperatures were measured daily, and the
severity of the clinical symptoms was evaluated based on a point
scale established by Lee et al. (2005) that independently consid-
ers attitude (04 points), respiratory rate (02 points), respiratory
distress (03 points), and cough (01 point) [24]. Sequential blood
samples were collected from all animals at 0, 3, 4, 5 and 7 days
Table 2
Immunizationregimensof peptides and Gp95N in piglets.
Group ID PBS Gp BT BT-Gp
Antigens PBS Gp96N aBCEsand TCEs peptides bBT peptides with Gp96N
Dose 2 ml per pig 0.5 mg/2 ml per pig (0.2 mg ofeachpeptidewithor without 0.5mg Gp96N)/2 ml perpig
a Themixture of two BCE peptides and eight TCE peptides shown in Table 1.b
The samepeptidesas the BT group mixed with Gp96N as an adjuvant.
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1840 C. Chen et al. / Vaccine31 (2013) 18381847
post-challenge (dpc). At 4 dpc, one piglet from each group was
euthanized for pathological examination.
All the animal research wasapprovedby the Beijing Association
for Science and Technology, with approval ID SYXK (Beijing) 2010-
0006 and SYXK(Beijing) 2006-0008, and complied with the Beijing
Laboratory Animal Welfare and Ethical Guidelines of the Beijing
Administration Committee of Laboratory Animals.
2.4. Immunofluorescence assay (IFA)
IFA was performed according to our previous report with slight
modifications [25]. Briefly, MARC-145 cells were infected with
PRRSV JXwn06 at a multiplicity of infection (MOI) of 1. Twenty-
four hours later, the cells were washed with PBS, fixed in 4%
paraformaldehyde, permeabilized with PBST (0.1% Triton X-100 in
PBS), and then incubated for 1h at 37C with 4% BSA in PBS. T he
cells were incubated with pig antisera (1:100) at 42dpi from the
fourgroups for 1 h at37 C. After washing five times with PBST, the
cells were incubated for 1h at 37 C with TRITC-conjugated anti-
swine IgG (1:100). After washing five times with PBST, the cells
were stained for nuclei by DAPI and observed using a Leica confocal
microscope.
2.5. Purification of PRRS virions
MARC-145 cells were infected with PRRSV JXwn06 isolate for
72h. The infected cells with cell cultures were frozen and thawed
three times and then centrifuged at 3000gat 4 C for half an hour.
The clarified culture supernatant (containing virus) was then con-
centrated by centrifugation (Beckman Coulter SW41 Ti rotor) at
100,000gat 4 C for 2 h. The precipitate (virions) was resuspended
in PBS. Subsequently, the virus was purified by ultracentrifuga-
tion through a 30% sucrose cushion using a SW41 Ti rotor at
100,000g for 2h, and the purified virions were resuspended in
PBS and heat-inactivated at 56C for 30 m in. At the same time,
the uninfected cells were purified with the same methods and
used as negative control (cell control). The concentration of the
purified virions (or cell control) was determined by the Bradford
assay using BSA as a standard, and the virions were stored at
80 C.
2.6. Monitoring antibodies by ELISA
BCE peptide-specific and PRRSV-specific antibody responses
weredetermined using an indirectELISA (iELISA)with BSA-coupled
BCE peptides or inactivated purified-PRRS virions (as described
above) as coating antigens. The 96-well ELISA plates were coated
overnight at 4 C with 1g/well coating antigens diluted in 100l
of 50mmol/l sodium carbonate buffer (pH 9.6). The plates were
washed three times withPBST (0.05% Tween-20in PBS)and blockedfor 1 h at 37 C with blocking buffer (3% BSA in PBS). Then, the
serum samples from piglets (including the negative sera collected
at 0 dpi) were diluted 1:100 in blocking buffer, added to each well
(100l/well) in duplicate, and incubated for 1h at 37 C. After
six washes, 100l of HRP-conjugated goat anti-swine IgG, diluted
1:5000 in blocking buffer, was added to each well and incubated at
37 C for 1h. After six washes, 100l of substrate solution (0.4M
tetramethylbenzidine and 1 mM H2O2 in 100mM acetate buffer,
pH 5.6) was added and incubated for 20min at room tempera-
ture in the dark, and the reaction was stopped by the addition
of 50l 2 M H2SO4 to each well. The absorbance was read at
450 nm (OD450) using an ELISA reader (Melgan). The OD450value
above 0.1 was determined as positive while under 0.1 as nega-
tive.
2.7. Lymphocyte proliferative response assay
Lymphocyte proliferation assays were performed using PBMCs
from the immunized piglets. The PBMCs were plated in 96-
well flat-bottom plates at 100l RMPI 1640 medium (containing
5% FBS, 100U /ml of penicillin and 100g /ml of streptomycin,
2105 cells/well) in triplicate. Subsequently, the inactivated
purified-PRRS virions (20g/ml, MARC-145 cells derived anti-
gens as a negative control) or each of the seven TCE peptide
(20g/ml, PBS as a negative control) was added and mixed.
Con A (5g/ml, Sigma) was used as a positive control. The
proliferative activity was measured according to a previously
described method with slight modifications [6]. Briefly, after a
72h incubation, 5mg/ml 3-(4,5-dimethylthylthiazol-2-yl)-5-(3-
carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS,
Sigma) was added to each well, and the plates were incubated for
anadditional 5 h, followed byadding 150l/well DMSOfor another
10min.At theendof theincubation,the plateswerereadat 570nm.
The stimulation index (SI) was calculated as the ratio of the aver-
age OD valueof the wellscontaining antigen-stimulatedcells to the
average OD value of negative control wells.
2.8. Cytokine assay
The levels of IL-4, IL-6, IL-10, IL-12, IFN-, and TNF- in the
sera and in the supernatant of the activated PBMCs were quan-
titatively determined using commercially available cytokine ELISA
Kits (R&D). PBMCs separated from the vaccinated piglets at 42dpi
were plated in 96-well flat-bottom plates at 100l RMPI 1640
medium (containing 5% FBS and antibiotics as described in Section
2.7, 2105 cells/well) in triplicate (fresh medium without cellswas
used as a blank control) and stimulated with purified inactivated-
PRRS virions (or MARC-145 cells derived antigens as a negative
control). After culturing for 72h, the supernatant from each well
was collected and immediately quantified by ELISA. Cytokines in
the sera at 0, 14, 28, and 42 days post primary immunization
were also examined according to the kits procedure. The cytokine
concentration of each sample was expressed as the mean of thetriplicates, while the cytokine quality of each group at the indi-
cated time point was calculated as the mean standard deviation
(SD) of four piglets.
2.9. Pathological examination
To estimate the severity of pathological lesions, the histologi-
cal pathology of lungs from the pigs at 4 dpc was determined as
described [26].
2.10. Statistical analysis
Statistical analyses and graphical presentation were performed
using GraphPad Prism 5.0software. All of the data are expressed asthe mean of four pigsSD. Differences in cell-mediated immune
responses, cytokine levels and viral titers at each sampling point
wereanalyzedusing the Wilconxon test.The clinicalsigns recorded
of pigs for each group were analyzed using the Fishers exact test.
P-values
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1842 C. Chen et al. / Vaccine31 (2013) 18381847
1001 sequences of Gp5, and 226 sequences of N were analyzed by
DNAStar andWeblogosoftware. As shown in Fig.1, eightof the epi-
topes were highly conserved, while Gp4-59B was more divergent.
Gp5-37B, Gp4-7T, and Gp4-170T displayed poor antigenic charac-
teristics using the Jameson-Wolf Antigenic analysis, while strong
antigenicity have been showed in Gp5-59B, Gp5-117T, Gp5-149T,
N-49T, N-63T, and N-104T. Thus, the consensus sequences of these
epitopes, together with PADRE, were chosen and synthesized with
KLH or BSA conjugated to Gp4-59B and Gp5-37B.
3.2. Humoral immune responses induced by BCE-peptides
First, IFA was used to determine whether the sera from the
BCE peptide-vaccinated piglets were PRRSV-specific. As shown in
Fig. 2A, JXwn06 PRRSV could be detected with pig sera against BCE
peptides (groups BT and BT-Gp). No staining was detected in the
infected cells with the sera from the pigs receiving PBS or Gp96N.
These results demonstrated that the two BCE peptides could stim-
ulate PRRSV-specific antibodies.
Next, the BSA-conjugated BCE peptides or the inactivated
purified-PRRS virions were used as coating antigens to monitor the
IgG responses by ELISA. A very low level of anti-BEC-peptides and
anti-PRRSV antibodies was detected in piglets from group BT after
three immunizations, while a low level of IgG was detected after
the second immunization and increased notably by the third vac-
cination in group BT-GP (Fig. 2B and C). Thus, Gp96N increased the
levels of BCE-specific (Fig. 2B) andPRRSV-specific(Fig.2C) IgGs. We
also determined thelevel ofNAs inthe sera from immunizedpiglets
by sera-virus neutralization assays. Unfortunately, no NA could be
detected in the sera of the immunized piglets.
3.3. TCE-induced cell-mediated immunity by TCE peptides
First, inactivated purified PRRS virions were used as a stimu-
latorto perform the lymphocyte proliferation assays. As shown in
Fig.3A, comparedto the PBSgroup ateachindicated time point,sig-
nificant PRRSV-specific lymphocyte proliferative responses were
exhibited in the PBMCs from the peptide-vaccinated piglets only
at 42dpi (P
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C. Chen et al. / Vaccine31 (2013) 18381847 1843
Fig. 4. Th1-type cytokine response bias by Gp96N in the peptides-vaccinated piglets. (A) PBMCs were collected and stimulated as mentioned in Fig. 3A. Six cytokinesweretested by quantitative ELISA from the supernatants of the PBMC cultures immediately after 72h of stimulation. (B) Cytokinesin the serum from the pigs of the PBS, Gp, BT,
and BT-Gp groups were detected by quantitative ELISA.Data areshownas themean SDof fourpigsfromeach group. *P
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Fig. 5. Detection of clinical sign and viremia of the pigs after challenge with HP-
PRRSV isolate JXwn06. (A) Rectal temperature of the pigs, with values expressed
as the mean SD. (B) Clinical sign scores of pigs per group are expressed as the
mean SD of allclinicalsignsrecordeddailypost challenge, includingattitude(03
points),respiratoryrate (02points),respiratorydistress (03points),and coughing
(01points). (C)The viremiaof thepigs isexpressed asthe viraltitersin seradetected
at 0 (before challenge), 3, 4, 5, and 7 dpc. The data are shown as the mean SD for
four orthreepigsper group.*P
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C. Chen et al. / Vaccine31 (2013) 18381847 1845
Fig. 6. Pathological examination.At 4 dpc, the lungs of the pigs were examined by HE staining in the PBS (A), Gp (B), BT (C), and BT-Gp (D) groups, as well as a healthy lung
from an uninfected pig (E).Pulmonaryalveoli were marked with hollow pentagon; solidtriangle exhibited thatfoci inflammatorycells infiltrated into the pulmonary alveoli,
and inflammatory cells infiltrated into the lumens as hollow triangle or near the vessel of the bronchus as solid arrow; hollow arrow represented the blood congestion in
lung. Scale bar: 200m.
is related to the cell-mediated immunity against PRRSV, was not
influenced by peptides, Gp96N, or peptides+ Gp96N (Fig. 4B). The
Th2 type cytokine IL-4 was down-regulated by peptides but not
Gp96N, and the combination of peptides+ Gp96N suppressed the
production of IL-4after the second immunization(Fig.4B). Further-
more, Gp96N inhibited the production of IL-10. It should be noted
that IL-10 is a potent immunosuppressive cytokine that can signif-
icantly inhibit production of several pro-inflammatory cytokinesand down-modulate APC activities, resulting in inhibition of innate
and adaptive immunity, particularly the cell-mediated immune
responses [46,47]. Concerning the inflammatory cytokines, Gp96N
or peptides did not alter the production of IL-6 (Fig. 4B). The other
innate anti-viral cytokine,TNF-, was significantlyup-regulatedby
Gp96N, but impaired by peptides because Fig.4A showed that pep-
tides might contribute to the down-regulated TNF- in group BT.
In addition, the TNF- level did not greatly increase until the third
immunization (at 42dpi in the BT-Gp group, Fig. 4B-iv). There may
be other unknown reasons that the TCE-peptides would delay the
up-regulation of TNF- by Gp96N. Taken together, Gp96N was an
effective Th1-biased adjuvant to improve the innate and adaptive
immune responses induced by the epitope-based vaccine, whichis
consistent with our previous report [25].
To determine the protective immune response induced by the
epitope-based peptides, the pigs were challenged with a high
dose of virulent HP-PRRSV isolate JXwn06. Temperature, clinical
symptoms, and viremia were monitored. Our results illustrate that
BCE+ TCE peptides with Gp96N as an adjuvant provided greater
protection than BCE +TCE peptides alone during the first 5 days
post HP-PRRSV infection. However, PRRSV infection was not fully
prevented in that all of the pigs in the BT-Gp group died at 7 dpcwitha hightiterof livePRRSVin their sera(Fig.5C) andserious clin-
ical symptoms (Fig. 5A and B, Fig. 6). One reason for this could be
that HP-PRRSV JXwn06 is a highly pathogenic isolate and causes
severe disease that leads to 100% mortality in 6-week-old pigs
within 513 days and 80% mortality in 12-week-old pigs [48]. In
this study, the piglets were 10 weeks old when challenged, which
mightbe related thedeath of the piglets. Additionally, the high dose
of HP-PRRSV JXwn06 challenge is another probable cause for the
death of the infected piglets. The viral dosage was 2105 TCID50in the present study, which is two times greater than the previous
report [48]. Based on the high level of mortality with 105 TCID50of HP-PRRSV infect, twice more dosage of virulent challenge virus
may cause much more serious mortality. This is the probable rea-
son for the 100% mortality at 7 dpc in the present study. Thus,
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