Accepted Manuscript Title: The amino acid residues at 102 and 104 in GP5 of porcine reproductive and respiratory syndrome virus regulate viral neutralization susceptibility to the porcine serum neutralizing antibody Author: Baochao Fan Xing Liu Juan Bai Tingjie Zhang Qiaoya Zhang Ping Jiang PII: DOI: Reference:
S0168-1702(15)00149-5 http://dx.doi.org/doi:10.1016/j.virusres.2015.04.015 VIRUS 96591
To appear in:
Virus Research
Received date: Revised date: Accepted date:
10-1-2015 8-4-2015 10-4-2015
Please cite this article as: Fan, B., Liu, X., Bai, J., Zhang, T., Zhang, Q., Jiang, P.,The amino acid residues at 102 and 104 in GP5 of porcine reproductive and respiratory syndrome virus regulate viral neutralization susceptibility to the porcine serum neutralizing antibody, Virus Research (2015), http://dx.doi.org/10.1016/j.virusres.2015.04.015 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Highlights
2
The NAb resistant PRRSV strains were generated under NAb pressure in vitro.
3
The 102 and 104 aa sites in GP5 regulate viral neutralization susceptibilities to porcine serum NAbs in MARC-145 and PAM cells.
5
The aa mutants Y102C and G104R also appear in wild type 2 PRRSV strains.
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Page 1 of 39
The amino acid residues at 102 and 104 in GP5 of porcine
8
reproductive and respiratory syndrome virus regulate viral
9
neutralization susceptibility to the porcine serum
10
neutralizing antibody
ip t
7
11
Baochao Fana*, Xing Liua*, Juan Baia, Tingjie Zhanga, Qiaoya Zhanga, Ping Jianga、b#
13
a. Key Laboratory of Animal Diseases Diagnostic and Immunology, Ministry of
14
Agriculture, College of Veterinary Medicine, Nanjing Agricultural University,
15
Nanjing 210095, China
16
b. Jiangsu Co-innovation Center for Prevention and Control of Important Animal
17
Infectious Diseases and Zoonoses, Yangzhou, China
M
an
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cr
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21 22 23 24
These authors contributed equally to this work.
#
Corresponding author:
pt
20
*
Dr. Ping Jiang
Ac ce
19
ed
18
College of Veterinary Medicine Nanjing Agricultural University, Nanjing 210095, P.R. China
25
Tel.: +86 25 84395504
26
Fax: + 86 25 84396640
27
[email protected]
28 29 2
Page 2 of 39
ABSTRACT
31
Porcine reproductive and respiratory syndrome virus (PRRSV) is mainly responsible
32
for the heavy economic losses in pig industry in the world. A number of neutralizing
33
epitopes have been identified in the viral structural proteins GP3, GP4, GP5 and M. In
34
this study, the important amino acid (aa) residues of HP-PRRSV strain BB affecting
35
neutralization susceptibility of antibody were examined using resistant strains
36
generated under neutralizing antibody (NAb) pressure in MARC-145 cells, reverse
37
genetic technique and virus neutralization assay. HP-PRRSV strain BB was passaged
38
under the pressure of porcine NAb serum in vitro. A resistant strain BB34s with 102
39
and 104 aa substitutions in GP5, which have been predicted to be the positive sites for
40
pressure selection (Delisle et al., 2012), was cloned and identified. To determine the
41
effect of the two aa residues on neutralization, 8 recombinant PRRSV strains were
42
generated, and neutralization assay results confirmed that the aa residues 102 and 104
43
in GP5 played an important role in NAbs against HP-PRRSV in MARC-145 cells and
44
porcine alveolar macrophages. Alignment of GP5 sequences revealed that the variant
45
aa residues at 102 and 104 were frequent among type 2 PRRSV strains. It may be
46
helpful for understanding the mechanism regulating the neutralization susceptibility of
47
PRRSV to the NAbs and monitoring the antigen variant strains in the field.
48
Key words: PRRSV; 102 and 104; neutralizing antibody.
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1. Introduction Porcine reproductive and respiratory syndrome virus (PRRSV) is a single-stranded, positive-sense
RNA
virus
that
belongs
to
the
Arteriviridae
family
of
55
the order Nidovirales (Gorbalenya et al., 2006). Based on genetic and antigenic
56
characteristics, two major genotypes of PRRSV, type 1 (European; prototype strain
57
Lelystad) and type 2 (North American; prototype strain VR-2332), have been
58
identified and share approximately 55–70% nucleotide identity (Andreyev et al., 1997;
59
Mateu et al., 2006; Meng et al., 1994; Nelsen et al., 1999). PRRSV is responsible for
60
reproduction problems in sows and boars, and respiratory problems in pigs of all ages
61
(Collins et al., 1992; Wensvoort et al., 1991). It is considered to be one of the most
62
economically important viruses in the swine industry worldwide (Neumann et al.,
63
2005; Pejsak et al., 1997). PRRSV genome is a positive, single-stranded, 5′-capped
64
and 3′-polyadenylated mRNA molecule, with a length of approximately 15,000
65
nucleotides (nt). It contains, in the direction 5′-3′, two large open reading frames
66
(ORFs), ORF1a and 1b, which encode the viral replicase and constitute approximately
67
three-quarters of the genome, and seven smaller ORFs, designated 2a, 2b and 3
68
through 7, which express structural proteins termed GP2a, E, GP3, GP4, GP5, M and
69
N, respectively (Meulenberg, 2000). An additional structural protein, GP5a, exists and
70
is encoded by an alternative ORF in the subgenomic viral mRNA encoding GP5
71
(Johnson et al., 2011).
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PRRSV can cause persistent infections in pigs. The NAbs in PRRSV-infected
73
animals are generated late and their titers remain low (reviewed by (Balasuriya and 4
Page 4 of 39
MacLachlan, 2004; Lopez and Osorio, 2004). The delayed or weak induction of NAbs
75
upon arterivirus infection has frequently been linked to GP5 glycosylation (Vu et al.,
76
2011). Passive transfer of NAbs to pigs prior to challenge with a homologous virulent
77
PRRSV strain results in complete protection of the pigs against infection,
78
demonstrating the important role of NAbs in protective immunity (Lopez et al., 2007;
79
Osorio et al., 2002). However, the role of anti-PRRSV NAbs for the virus diversity
80
and evolution has not been understood completely.
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The genomic variation sometimes results in the emergence of new PRRSV
82
populations (Allende et al., 2000; Chang et al., 2002; Rowland et al., 1999). It is
83
expected that the adaptive immune response of the host will act as an important
84
source of selective pressure in the evolutionary process of the virus (Al-Gelban, 2004;
85
Domingo et al., 2001). Several studies have been published on the examination of
86
PRRSV sequence variability followed the selective pressure on PRRSV genes during
87
infection in vivo. But they did not reveal a correlation between regions under high
88
positive selection and the location of neutralizing B-cell epitopes (Allende et al., 2000;
89
Chang et al., 2002; Goldberg et al., 2003). Costers et al (2010) isolated the antibody
90
escape variants from the vaccinated pigs and reveled that NAbs in pigs was a driving
91
force in the rapid evolution of the neutralizing epitope on GP4 of type 1 PRRSV
92
strains (Costers et al., 2010a; Costers et al., 2010b).
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GP5 is the main target for NAbs in type 2 PRRSV strains (Gonin et al., 1999;
94
Ostrowski et al., 2002; Plagemann et al., 2002). A neutralizing epitope (aa 37 to 45:
95
SHLQLIYNL) has also been identified on GP5 (Plagemann, 2004). A recent report
5
Page 5 of 39
argued that the major envelope protein surface epitopes were disassociated with the
97
virus neutralization (Li and Murtaugh, 2012). Meanwhile, it was suggested that the aa
98
sites 102 and 104 in GP5 were likely positive sites under some selective pressure by
99
host immune cells based on a large dataset of 1301 sequences (1998–2009) analysis
100
(Delisle et al., 2012). In this study, a type 2 highly pathogenic PRRSV (HP-PRRSV)
101
strain BB0907 (tenth passage, termed BB) was passaged in MARC-145 cells in the
102
presence of porcine serum antibodies against this PRRSV strain, and five resistant
103
variants were generated. The sequencing results showed that BB30s and BB34s had
104
aa substitutions at 102 and 104 in GP5 gene. Then 8 recombinant PRRSV strains
105
containing these mutations were generated by site-directed mutagenesis using BB and
106
BB34s infectious cDNA clones. It was found that the aa residues at 102 and 104 in
107
GP5 of PRRSV played important role in escaping from the NAbs against HP-PRRSV.
108
Our data may provide important information for understanding the molecular
109
mechanism regulating the neutralization susceptibility of PRRSV to NAbs and may be
110
helpful for monitoring the antigen variant strains in the field.
111
2. Materials and methods
112
2.1. Cells, viruses and NAb serums
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MARC-145 cells were maintained in Dulbecco's Modified Eagle's medium
114
(DMEM, GIBCO, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum
115
(FBS, GIBCO) containing 100 U penicillin/ml and 100 μg streptomycin/ml at 37°C
116
with 5% CO2. 6
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The HP-PRRSV isolate BB0907 (GenBank no. HQ315835, tenth passage by using
118
MARC-145 cells，termed BB) used in this study was isolated in Guangxi Province,
119
China, in 2009. The recombinant PRRSV strains (rBB, rBB/GP5s, rBB34s,
120
rBB34s/GP5, rBB/GP5(Y102C), rBB/GP5(G104R), rBB/GP5s-R and rBB34s/GP5-R)
121
were constructed by site-directed mutagenesis of the aa residues according to
122
conventional methods (Fig. 1). The titers of the viral stocks were determined by
123
measuring their cytopathic effect (CPE) in MARC-145 cells.
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Five anti-HP-PRRSV neutralizing serums were prepared from five 45-day-old
125
piglets free of PRRSV by inoculated with low dose (104 TCID50) of HP-PRRSV strain
126
BB by 2 times with interval 28 days. At 70 days post inoculation, the bloods were
127
collected and the serums were isolated and named N1, N2, N3, N4 and N5,
128
respectively. The neutralizing antibody (NA) titers of these serums against
129
HP-PRRSV BB were 1: 34-1:48.
130
2.2. Isolation of antibody-resistant variants
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To select variants resistant to NAb against HP-PRRSV, PRRSV strain BB (100
132
TCID50) was mixed with a 100-fold dilution of the anti-PRRSV antibody and
133
incubated at 37°C for 1 h before infecting MARC-145 cell monolayers. The virus
134
arising from the cells showing a CPE was used for the subsequent passage. After
135
passaged by 5-30 times under the antibody pressure, five NAb-resistant variant clones
136
were selected by using plaque tests. The titers of the resistant virus were 106–108
137
TCID50/mL.
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Page 7 of 39
138
2.3. Sequencing of resistant variants Viral RNA was purified from culture supernatants taken from cells showing a CPE
140
using the QIAmp viral RNA Mini Kit (Qiagen, Hilden, Germany), and reverse
141
transcriptase PCR (RT-PCR) was performed to generate cDNA, according to the
142
manufacturer’s protocol, using the SuperScript III First-Strand Synthesis Kit
143
(Invitrogen, Carlsbad, CA, USA). The complete genome of the virus was divided into
144
seven overlapping fragments for amplification, and the 5′ and 3′ termini of the
145
genomic were synthesized using rapid amplification of the cDNA ends (RACE). The
146
six structural protein genes (GP2, GP3, GP4, GP5, M and N) were amplified using the
147
Phanta Super Fidelity DNA Polymerase (Vazyme, China) with the special primers.
148
2.4. Serum neutralization assay
ed
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The NA titers of the antibody serums against the parent and mutant HP-PRRSVs
150
were assayed in MARC-145 cells as described previously (Jiang et al., 2006) with
151
minor modifications. The viruses were diluted to a concentration of 100 TCID50 per
152
50 μl (103.3 TCID50/ml) in DMEM supplemented with 2% FBS. Serial dilutions of the
153
neutralizing serums were mixed with each of the viruses and incubated at 37°C for 1 h.
154
The mixtures (100 μl/well) were transferred to MARC-145 monolayers in 96-well
155
plates and incubated for an additional 4 days at 37°C with 5% CO2. Every serum
156
dilution contained four replicate wells. Cells were then examined for CPE. NA titers
157
were expressed as the reciprocal of the highest dilution that completely inhibited the
158
appearance of the CPE.
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Page 8 of 39
In addition, the NA titers of the antibody serums against PRRSVs were detected
160
in porcine alveolar macrophages (PAMs) as previously description (Vanhee et al.,
161
2010) with minor modifications. In brief, PAM cells were prepared from 4-week-old
162
piglets free of PRRSV by lung lavage and adjusted to 5×106/mL with RPMI-1640
163
(GIBCO) medium containing 10% fetal bovine serum, 100 units/mL of penicillin, and
164
100 μg/mL of streptomycin. The cells were added to 96-well culture flasks (Costar,
165
Corning Incorporated, NY) and incubated for 6 h at 37°C in a humidified
166
compartment to allow cells to adhere to flasks. Two-fold serial dilutions of serum N4
167
in RPMI-1640 were mixed with equal volumes of 100 TCID50 PRRSV strains, and
168
incubated for 1 h at 37°C and transferred to a 96-well plate (100 μl/well) with PAMs.
169
The inoculum was removed after 1h and replaced by medium, after which the cells
170
were further incubated for another 10 h. The cells were fixed and stained with mAb
171
against the N protein of PRRSV and FITC-conjugated goat anti-mouse IgG (BOSTER,
172
China). The NA titers were determined as the reciprocal of the highest dilution that
173
resulted in more than 90% reduction of infected cells.
174
2.5. Construction of infectious cDNA clones of PRRSVs
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The full-length PRRSV genome was amplified using the five primer pairs listed in
176
Table S1. A recombinant plasmid (pCMV-BB) containing the full-length cDNA of the
177
BB was constructed as shown in Fig. 1. To introduce the NAb-resistant mutations of
178
the structural protein GP5 into PRRSV infectious cDNA clone pCMV-BB,
179
site-directed mutagenesis was employed using the QuikChange® II XL Site-Directed
180
Mutagenesis kit (Stratagene, La Jolla, CA, USA) according to the manufacturer's 9
Page 9 of 39
181
recommendations. First, fragment D, which encodes the structural proteins, was
182
amplified using pCMV-BB as template and cloned into the pEASY-Simple Blunt
183
vector (Beijing TransGen Biotech Co., Ltd., Beijing, China) using AscI and SpeI
184
restriction
185
pEASY-BB-D, which was used as the intermediate plasmid. Second, the site-directed
186
mutagenesis constructs were prepared using pEASY-BB-D as template. Briefly, the
187
oligonucleotide primers were designed such that a foreign insertion sequence was
188
incorporated at the 5′ end or in the middle of the primer, leaving at least ten
189
nucleotides at the 3′ end that matched the template sequence. PCRs were run
190
according to the instructions of the QuickChange mutagenesis kit using circular
191
plasmid DNA as the template. The plasmid template was eliminated by DpnI
192
digestion (New England Biolabs), followed by transformation of the digested PCR
193
mixtures into Top10 competent cells (Invitrogen). The intermediate plasmids were
194
screened and verified by restriction enzyme mapping and nucleotide sequencing.
195
Third, the mutations in pEASY-BB-D and pCMV-BB were digested with AscI/SpeI,
196
and fragment D in pCMV-BB was replaced by the analogous fragments derived from
197
pEASY-BB-D, and the full-length mutant clones from pCMV-BB were obtained.
198
Meanwhile, the plasmid pCMV-BB34s and its site-directed mutation plasmids were
199
also constructed according the above methods (Fig. 1). Besides, two different reverse
200
mutants that were constructed from the full-length infectious cDNA clones
201
pCMV-BB/GP5s and pCMV-BB34s/GP5 by the site-directed mutagenesis described
202
above, respectively (Fig. 1). All the recombinant viruses and the aa mutations were
(New
England
Biolabs,
USA),
thereby
yielding
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endonucleases
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Page 10 of 39
203
summarized in Table 1.
204
2.6. Rescue of recombinant viruses Plasmids carrying a full-length PRRSV cDNA were individually transfected into
206
MARC-145 cells using Lipofectamine 2000 (Invitrogen) according to the
207
manufacturer’s instructions. Four days after transfection, the rescues of infectious
208
viruses were obtained and cloned by the plaque assay. The mutations in the rescued
209
viruses were confirmed by RT-PCR and sequencing.
210
2.7. Viral plaque assay
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MARC-145 cells in 12-well plates were inoculated with 100 μl of tenfold serially
212
diluted PRRSV. After 1 h adsorption at 37°C, cell monolayers were washed with
213
phosphate-buffered saline (PBS) and overlaid with 1% low melting agarose in DMEM
214
(Invitrogen) containing 2% FBS. After the gel overlay solidified, the plates were
215
inverted (top side down) and placed into an incubator at 37°C with 5% CO2. At 4 days
216
post-infection (dpi), plaques were visualized by crystal violet staining.
217
2.8. Antibody binding analyses
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M
211
Parent and recombinant mutant PRRSVs were purified by ultracentrifugation and
219
diluted in coating buffer to a final concentration 5 g/ml. The antigens were added in
220
triplicate to 96-well flat-bottomed enzyme-linked immunosorbent assay (ELISA)
221
plates. After incubation at 4°C for at least 16 h, wells were blocked with PBS-5% FBS
222
for 1 h at room temperature. A non-saturating concentration of the serum antibody
223
(diluted 1:100), which was in the linear portion of the antibody titration curve 11
Page 11 of 39
determined previously, was added to each well, serially diluted twofold, and incubated
225
with the virus-coated plates for 1 h at room temperature. After washing, anti-pig
226
horseradish peroxidase (HRP)-conjugated antibody was added to the plates, and they
227
were incubated for 1 h at room temperature. After another round of washing,
228
3,3’,5,5’-tetramethylbenzidine (TMB) substrate (KPL Biomedical, Gaithersburg, MD)
229
was added, and 2 M H2SO4 was used to stop the reaction. The amount of HRP product
230
was determined using a plate reader at 450 nm. The control plates were coated with
231
ultracentrifuged non-infected MARC-145 cell lysates.
232
2.9. Western blot
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In order to evaluate the consistence of PRRSV antigens binding in the wells, the
234
antigens were lysed from the coated 96-well ELISA plate by using RIPA Lysis Buffer
235
(Beyotime, China). Briefly, 100μl lysis buffer was added into one coated well. After 5
236
minutes, the lysate samples were obtained and added to another coated well. A total of
237
12 wells (for one strain antigen) were lysed and obtained as one sample. Then all the
238
PRRSV strains antigen samples from the coated plate and the original purified
239
antigens were separated by 12% SDS-PAGE, and transferred onto nitrocellulose filter
240
membrane (PALL, New York, USA). The membranes were incubated with mAb N
241
(made in our laboratory) or GP5 (provided by Dr GZ. Tong, Shanghai Veterinary
242
Research Institute, China) as the primary antibody. After the membranes were rinsed
243
with PBS, the membrane was treated with goat anti-mouse IgG-HRP (BOSTER,
244
China) as the secondary antibody. The proteins were visualized by scanning the
245
membranes with the Tanon 5200 chemiluminescence imaging system (Tanon, China).
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Page 12 of 39
246
2.10. Growth curves of viruses To determine viral one-step growth curves, PRRSV mutants (105 TCID50) were
248
inoculated into sub-confluent MARC-145 cells or PAMs in six-well plates. Then, 200
249
μl of the supernatants of the infected cells was collected and replenished with the
250
same volume of fresh medium at 12, 24, 36, 48, and 72 h post-infection (hpi), and
251
stored at −70 °C for virus titration. The virus titers for each time point were
252
determined in MARC-145 cells by TCID50.
253
2.11. Statistical analysis
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All data were analyzed using GraphPad Prism (Version 5.03, San Diego,
255
California) software. Differences among all groups were examined using one-way
256
analysis of variance (ANOVA), followed by Tukey’s tests. Differences between two
257
groups were assessed using unpaired two-tailed t-tests. Differences were considered
258
significant if P was < 0.05.
259
3. Results
260
3.1. Generation of resistant variants against NAb to PRRSV
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M
254
Five different NAb serums were prepared from the PRRSV-free piglets infected
262
with PRRSV strain BB. The NA titers of the serums against this strain were 1:34-1:48.
263
We used the N4 NAb serum that had the highest NA titer (1:48) to generate the
264
resistant variants from the parental virus BB.
265
After 5-34 passages or purification of PRRSV BB in MARC-145 cells in the
13
Page 13 of 39
presence of N4 NAb to PRRSV, five resistant strains BB5s, BB10s, BB20s, BB30s
267
and BB34s, which could replicated in MARC-145 under pressure of NAb to PRRSV,
268
were obtained. As shown in Fig. 2, the plaque morphologies of BB30s and BB34s
269
were larger than that of the parental strain BB in size (Fig. 2A). Neutralization assays
270
results showed that BB10s and BB20s had a similar NA titers that were significantly
271
lower against that of BB (P
S0168-1702(15)00149-5 http://dx.doi.org/doi:10.1016/j.virusres.2015.04.015 VIRUS 96591
To appear in:
Virus Research
Received date: Revised date: Accepted date:
10-1-2015 8-4-2015 10-4-2015
Please cite this article as: Fan, B., Liu, X., Bai, J., Zhang, T., Zhang, Q., Jiang, P.,The amino acid residues at 102 and 104 in GP5 of porcine reproductive and respiratory syndrome virus regulate viral neutralization susceptibility to the porcine serum neutralizing antibody, Virus Research (2015), http://dx.doi.org/10.1016/j.virusres.2015.04.015 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Highlights
2
The NAb resistant PRRSV strains were generated under NAb pressure in vitro.
3
The 102 and 104 aa sites in GP5 regulate viral neutralization susceptibilities to porcine serum NAbs in MARC-145 and PAM cells.
5
The aa mutants Y102C and G104R also appear in wild type 2 PRRSV strains.
ip t
4
cr
6
Ac ce
pt
ed
M
an
us
7
1
Page 1 of 39
The amino acid residues at 102 and 104 in GP5 of porcine
8
reproductive and respiratory syndrome virus regulate viral
9
neutralization susceptibility to the porcine serum
10
neutralizing antibody
ip t
7
11
Baochao Fana*, Xing Liua*, Juan Baia, Tingjie Zhanga, Qiaoya Zhanga, Ping Jianga、b#
13
a. Key Laboratory of Animal Diseases Diagnostic and Immunology, Ministry of
14
Agriculture, College of Veterinary Medicine, Nanjing Agricultural University,
15
Nanjing 210095, China
16
b. Jiangsu Co-innovation Center for Prevention and Control of Important Animal
17
Infectious Diseases and Zoonoses, Yangzhou, China
M
an
us
cr
12
21 22 23 24
These authors contributed equally to this work.
#
Corresponding author:
pt
20
*
Dr. Ping Jiang
Ac ce
19
ed
18
College of Veterinary Medicine Nanjing Agricultural University, Nanjing 210095, P.R. China
25
Tel.: +86 25 84395504
26
Fax: + 86 25 84396640
27
[email protected]
28 29 2
Page 2 of 39
ABSTRACT
31
Porcine reproductive and respiratory syndrome virus (PRRSV) is mainly responsible
32
for the heavy economic losses in pig industry in the world. A number of neutralizing
33
epitopes have been identified in the viral structural proteins GP3, GP4, GP5 and M. In
34
this study, the important amino acid (aa) residues of HP-PRRSV strain BB affecting
35
neutralization susceptibility of antibody were examined using resistant strains
36
generated under neutralizing antibody (NAb) pressure in MARC-145 cells, reverse
37
genetic technique and virus neutralization assay. HP-PRRSV strain BB was passaged
38
under the pressure of porcine NAb serum in vitro. A resistant strain BB34s with 102
39
and 104 aa substitutions in GP5, which have been predicted to be the positive sites for
40
pressure selection (Delisle et al., 2012), was cloned and identified. To determine the
41
effect of the two aa residues on neutralization, 8 recombinant PRRSV strains were
42
generated, and neutralization assay results confirmed that the aa residues 102 and 104
43
in GP5 played an important role in NAbs against HP-PRRSV in MARC-145 cells and
44
porcine alveolar macrophages. Alignment of GP5 sequences revealed that the variant
45
aa residues at 102 and 104 were frequent among type 2 PRRSV strains. It may be
46
helpful for understanding the mechanism regulating the neutralization susceptibility of
47
PRRSV to the NAbs and monitoring the antigen variant strains in the field.
48
Key words: PRRSV; 102 and 104; neutralizing antibody.
Ac ce
pt
ed
M
an
us
cr
ip t
30
49 50 51 3
Page 3 of 39
52
53
1. Introduction Porcine reproductive and respiratory syndrome virus (PRRSV) is a single-stranded, positive-sense
RNA
virus
that
belongs
to
the
Arteriviridae
family
of
55
the order Nidovirales (Gorbalenya et al., 2006). Based on genetic and antigenic
56
characteristics, two major genotypes of PRRSV, type 1 (European; prototype strain
57
Lelystad) and type 2 (North American; prototype strain VR-2332), have been
58
identified and share approximately 55–70% nucleotide identity (Andreyev et al., 1997;
59
Mateu et al., 2006; Meng et al., 1994; Nelsen et al., 1999). PRRSV is responsible for
60
reproduction problems in sows and boars, and respiratory problems in pigs of all ages
61
(Collins et al., 1992; Wensvoort et al., 1991). It is considered to be one of the most
62
economically important viruses in the swine industry worldwide (Neumann et al.,
63
2005; Pejsak et al., 1997). PRRSV genome is a positive, single-stranded, 5′-capped
64
and 3′-polyadenylated mRNA molecule, with a length of approximately 15,000
65
nucleotides (nt). It contains, in the direction 5′-3′, two large open reading frames
66
(ORFs), ORF1a and 1b, which encode the viral replicase and constitute approximately
67
three-quarters of the genome, and seven smaller ORFs, designated 2a, 2b and 3
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through 7, which express structural proteins termed GP2a, E, GP3, GP4, GP5, M and
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N, respectively (Meulenberg, 2000). An additional structural protein, GP5a, exists and
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is encoded by an alternative ORF in the subgenomic viral mRNA encoding GP5
71
(Johnson et al., 2011).
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PRRSV can cause persistent infections in pigs. The NAbs in PRRSV-infected
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animals are generated late and their titers remain low (reviewed by (Balasuriya and 4
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MacLachlan, 2004; Lopez and Osorio, 2004). The delayed or weak induction of NAbs
75
upon arterivirus infection has frequently been linked to GP5 glycosylation (Vu et al.,
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2011). Passive transfer of NAbs to pigs prior to challenge with a homologous virulent
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PRRSV strain results in complete protection of the pigs against infection,
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demonstrating the important role of NAbs in protective immunity (Lopez et al., 2007;
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Osorio et al., 2002). However, the role of anti-PRRSV NAbs for the virus diversity
80
and evolution has not been understood completely.
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The genomic variation sometimes results in the emergence of new PRRSV
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populations (Allende et al., 2000; Chang et al., 2002; Rowland et al., 1999). It is
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expected that the adaptive immune response of the host will act as an important
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source of selective pressure in the evolutionary process of the virus (Al-Gelban, 2004;
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Domingo et al., 2001). Several studies have been published on the examination of
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PRRSV sequence variability followed the selective pressure on PRRSV genes during
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infection in vivo. But they did not reveal a correlation between regions under high
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positive selection and the location of neutralizing B-cell epitopes (Allende et al., 2000;
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Chang et al., 2002; Goldberg et al., 2003). Costers et al (2010) isolated the antibody
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escape variants from the vaccinated pigs and reveled that NAbs in pigs was a driving
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force in the rapid evolution of the neutralizing epitope on GP4 of type 1 PRRSV
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strains (Costers et al., 2010a; Costers et al., 2010b).
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GP5 is the main target for NAbs in type 2 PRRSV strains (Gonin et al., 1999;
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Ostrowski et al., 2002; Plagemann et al., 2002). A neutralizing epitope (aa 37 to 45:
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SHLQLIYNL) has also been identified on GP5 (Plagemann, 2004). A recent report
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argued that the major envelope protein surface epitopes were disassociated with the
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virus neutralization (Li and Murtaugh, 2012). Meanwhile, it was suggested that the aa
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sites 102 and 104 in GP5 were likely positive sites under some selective pressure by
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host immune cells based on a large dataset of 1301 sequences (1998–2009) analysis
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(Delisle et al., 2012). In this study, a type 2 highly pathogenic PRRSV (HP-PRRSV)
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strain BB0907 (tenth passage, termed BB) was passaged in MARC-145 cells in the
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presence of porcine serum antibodies against this PRRSV strain, and five resistant
103
variants were generated. The sequencing results showed that BB30s and BB34s had
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aa substitutions at 102 and 104 in GP5 gene. Then 8 recombinant PRRSV strains
105
containing these mutations were generated by site-directed mutagenesis using BB and
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BB34s infectious cDNA clones. It was found that the aa residues at 102 and 104 in
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GP5 of PRRSV played important role in escaping from the NAbs against HP-PRRSV.
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Our data may provide important information for understanding the molecular
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mechanism regulating the neutralization susceptibility of PRRSV to NAbs and may be
110
helpful for monitoring the antigen variant strains in the field.
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2. Materials and methods
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2.1. Cells, viruses and NAb serums
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MARC-145 cells were maintained in Dulbecco's Modified Eagle's medium
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(DMEM, GIBCO, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum
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(FBS, GIBCO) containing 100 U penicillin/ml and 100 μg streptomycin/ml at 37°C
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with 5% CO2. 6
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The HP-PRRSV isolate BB0907 (GenBank no. HQ315835, tenth passage by using
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MARC-145 cells，termed BB) used in this study was isolated in Guangxi Province,
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China, in 2009. The recombinant PRRSV strains (rBB, rBB/GP5s, rBB34s,
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rBB34s/GP5, rBB/GP5(Y102C), rBB/GP5(G104R), rBB/GP5s-R and rBB34s/GP5-R)
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were constructed by site-directed mutagenesis of the aa residues according to
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conventional methods (Fig. 1). The titers of the viral stocks were determined by
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measuring their cytopathic effect (CPE) in MARC-145 cells.
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Five anti-HP-PRRSV neutralizing serums were prepared from five 45-day-old
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piglets free of PRRSV by inoculated with low dose (104 TCID50) of HP-PRRSV strain
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BB by 2 times with interval 28 days. At 70 days post inoculation, the bloods were
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collected and the serums were isolated and named N1, N2, N3, N4 and N5,
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respectively. The neutralizing antibody (NA) titers of these serums against
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HP-PRRSV BB were 1: 34-1:48.
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2.2. Isolation of antibody-resistant variants
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To select variants resistant to NAb against HP-PRRSV, PRRSV strain BB (100
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TCID50) was mixed with a 100-fold dilution of the anti-PRRSV antibody and
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incubated at 37°C for 1 h before infecting MARC-145 cell monolayers. The virus
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arising from the cells showing a CPE was used for the subsequent passage. After
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passaged by 5-30 times under the antibody pressure, five NAb-resistant variant clones
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were selected by using plaque tests. The titers of the resistant virus were 106–108
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TCID50/mL.
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2.3. Sequencing of resistant variants Viral RNA was purified from culture supernatants taken from cells showing a CPE
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using the QIAmp viral RNA Mini Kit (Qiagen, Hilden, Germany), and reverse
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transcriptase PCR (RT-PCR) was performed to generate cDNA, according to the
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manufacturer’s protocol, using the SuperScript III First-Strand Synthesis Kit
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(Invitrogen, Carlsbad, CA, USA). The complete genome of the virus was divided into
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seven overlapping fragments for amplification, and the 5′ and 3′ termini of the
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genomic were synthesized using rapid amplification of the cDNA ends (RACE). The
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six structural protein genes (GP2, GP3, GP4, GP5, M and N) were amplified using the
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Phanta Super Fidelity DNA Polymerase (Vazyme, China) with the special primers.
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2.4. Serum neutralization assay
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The NA titers of the antibody serums against the parent and mutant HP-PRRSVs
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were assayed in MARC-145 cells as described previously (Jiang et al., 2006) with
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minor modifications. The viruses were diluted to a concentration of 100 TCID50 per
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50 μl (103.3 TCID50/ml) in DMEM supplemented with 2% FBS. Serial dilutions of the
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neutralizing serums were mixed with each of the viruses and incubated at 37°C for 1 h.
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The mixtures (100 μl/well) were transferred to MARC-145 monolayers in 96-well
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plates and incubated for an additional 4 days at 37°C with 5% CO2. Every serum
156
dilution contained four replicate wells. Cells were then examined for CPE. NA titers
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were expressed as the reciprocal of the highest dilution that completely inhibited the
158
appearance of the CPE.
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In addition, the NA titers of the antibody serums against PRRSVs were detected
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in porcine alveolar macrophages (PAMs) as previously description (Vanhee et al.,
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2010) with minor modifications. In brief, PAM cells were prepared from 4-week-old
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piglets free of PRRSV by lung lavage and adjusted to 5×106/mL with RPMI-1640
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(GIBCO) medium containing 10% fetal bovine serum, 100 units/mL of penicillin, and
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100 μg/mL of streptomycin. The cells were added to 96-well culture flasks (Costar,
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Corning Incorporated, NY) and incubated for 6 h at 37°C in a humidified
166
compartment to allow cells to adhere to flasks. Two-fold serial dilutions of serum N4
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in RPMI-1640 were mixed with equal volumes of 100 TCID50 PRRSV strains, and
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incubated for 1 h at 37°C and transferred to a 96-well plate (100 μl/well) with PAMs.
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The inoculum was removed after 1h and replaced by medium, after which the cells
170
were further incubated for another 10 h. The cells were fixed and stained with mAb
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against the N protein of PRRSV and FITC-conjugated goat anti-mouse IgG (BOSTER,
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China). The NA titers were determined as the reciprocal of the highest dilution that
173
resulted in more than 90% reduction of infected cells.
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2.5. Construction of infectious cDNA clones of PRRSVs
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The full-length PRRSV genome was amplified using the five primer pairs listed in
176
Table S1. A recombinant plasmid (pCMV-BB) containing the full-length cDNA of the
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BB was constructed as shown in Fig. 1. To introduce the NAb-resistant mutations of
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the structural protein GP5 into PRRSV infectious cDNA clone pCMV-BB,
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site-directed mutagenesis was employed using the QuikChange® II XL Site-Directed
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Mutagenesis kit (Stratagene, La Jolla, CA, USA) according to the manufacturer's 9
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recommendations. First, fragment D, which encodes the structural proteins, was
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amplified using pCMV-BB as template and cloned into the pEASY-Simple Blunt
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vector (Beijing TransGen Biotech Co., Ltd., Beijing, China) using AscI and SpeI
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restriction
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pEASY-BB-D, which was used as the intermediate plasmid. Second, the site-directed
186
mutagenesis constructs were prepared using pEASY-BB-D as template. Briefly, the
187
oligonucleotide primers were designed such that a foreign insertion sequence was
188
incorporated at the 5′ end or in the middle of the primer, leaving at least ten
189
nucleotides at the 3′ end that matched the template sequence. PCRs were run
190
according to the instructions of the QuickChange mutagenesis kit using circular
191
plasmid DNA as the template. The plasmid template was eliminated by DpnI
192
digestion (New England Biolabs), followed by transformation of the digested PCR
193
mixtures into Top10 competent cells (Invitrogen). The intermediate plasmids were
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screened and verified by restriction enzyme mapping and nucleotide sequencing.
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Third, the mutations in pEASY-BB-D and pCMV-BB were digested with AscI/SpeI,
196
and fragment D in pCMV-BB was replaced by the analogous fragments derived from
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pEASY-BB-D, and the full-length mutant clones from pCMV-BB were obtained.
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Meanwhile, the plasmid pCMV-BB34s and its site-directed mutation plasmids were
199
also constructed according the above methods (Fig. 1). Besides, two different reverse
200
mutants that were constructed from the full-length infectious cDNA clones
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pCMV-BB/GP5s and pCMV-BB34s/GP5 by the site-directed mutagenesis described
202
above, respectively (Fig. 1). All the recombinant viruses and the aa mutations were
(New
England
Biolabs,
USA),
thereby
yielding
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endonucleases
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summarized in Table 1.
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2.6. Rescue of recombinant viruses Plasmids carrying a full-length PRRSV cDNA were individually transfected into
206
MARC-145 cells using Lipofectamine 2000 (Invitrogen) according to the
207
manufacturer’s instructions. Four days after transfection, the rescues of infectious
208
viruses were obtained and cloned by the plaque assay. The mutations in the rescued
209
viruses were confirmed by RT-PCR and sequencing.
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2.7. Viral plaque assay
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MARC-145 cells in 12-well plates were inoculated with 100 μl of tenfold serially
212
diluted PRRSV. After 1 h adsorption at 37°C, cell monolayers were washed with
213
phosphate-buffered saline (PBS) and overlaid with 1% low melting agarose in DMEM
214
(Invitrogen) containing 2% FBS. After the gel overlay solidified, the plates were
215
inverted (top side down) and placed into an incubator at 37°C with 5% CO2. At 4 days
216
post-infection (dpi), plaques were visualized by crystal violet staining.
217
2.8. Antibody binding analyses
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Parent and recombinant mutant PRRSVs were purified by ultracentrifugation and
219
diluted in coating buffer to a final concentration 5 g/ml. The antigens were added in
220
triplicate to 96-well flat-bottomed enzyme-linked immunosorbent assay (ELISA)
221
plates. After incubation at 4°C for at least 16 h, wells were blocked with PBS-5% FBS
222
for 1 h at room temperature. A non-saturating concentration of the serum antibody
223
(diluted 1:100), which was in the linear portion of the antibody titration curve 11
Page 11 of 39
determined previously, was added to each well, serially diluted twofold, and incubated
225
with the virus-coated plates for 1 h at room temperature. After washing, anti-pig
226
horseradish peroxidase (HRP)-conjugated antibody was added to the plates, and they
227
were incubated for 1 h at room temperature. After another round of washing,
228
3,3’,5,5’-tetramethylbenzidine (TMB) substrate (KPL Biomedical, Gaithersburg, MD)
229
was added, and 2 M H2SO4 was used to stop the reaction. The amount of HRP product
230
was determined using a plate reader at 450 nm. The control plates were coated with
231
ultracentrifuged non-infected MARC-145 cell lysates.
232
2.9. Western blot
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In order to evaluate the consistence of PRRSV antigens binding in the wells, the
234
antigens were lysed from the coated 96-well ELISA plate by using RIPA Lysis Buffer
235
(Beyotime, China). Briefly, 100μl lysis buffer was added into one coated well. After 5
236
minutes, the lysate samples were obtained and added to another coated well. A total of
237
12 wells (for one strain antigen) were lysed and obtained as one sample. Then all the
238
PRRSV strains antigen samples from the coated plate and the original purified
239
antigens were separated by 12% SDS-PAGE, and transferred onto nitrocellulose filter
240
membrane (PALL, New York, USA). The membranes were incubated with mAb N
241
(made in our laboratory) or GP5 (provided by Dr GZ. Tong, Shanghai Veterinary
242
Research Institute, China) as the primary antibody. After the membranes were rinsed
243
with PBS, the membrane was treated with goat anti-mouse IgG-HRP (BOSTER,
244
China) as the secondary antibody. The proteins were visualized by scanning the
245
membranes with the Tanon 5200 chemiluminescence imaging system (Tanon, China).
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2.10. Growth curves of viruses To determine viral one-step growth curves, PRRSV mutants (105 TCID50) were
248
inoculated into sub-confluent MARC-145 cells or PAMs in six-well plates. Then, 200
249
μl of the supernatants of the infected cells was collected and replenished with the
250
same volume of fresh medium at 12, 24, 36, 48, and 72 h post-infection (hpi), and
251
stored at −70 °C for virus titration. The virus titers for each time point were
252
determined in MARC-145 cells by TCID50.
253
2.11. Statistical analysis
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All data were analyzed using GraphPad Prism (Version 5.03, San Diego,
255
California) software. Differences among all groups were examined using one-way
256
analysis of variance (ANOVA), followed by Tukey’s tests. Differences between two
257
groups were assessed using unpaired two-tailed t-tests. Differences were considered
258
significant if P was < 0.05.
259
3. Results
260
3.1. Generation of resistant variants against NAb to PRRSV
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Five different NAb serums were prepared from the PRRSV-free piglets infected
262
with PRRSV strain BB. The NA titers of the serums against this strain were 1:34-1:48.
263
We used the N4 NAb serum that had the highest NA titer (1:48) to generate the
264
resistant variants from the parental virus BB.
265
After 5-34 passages or purification of PRRSV BB in MARC-145 cells in the
13
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presence of N4 NAb to PRRSV, five resistant strains BB5s, BB10s, BB20s, BB30s
267
and BB34s, which could replicated in MARC-145 under pressure of NAb to PRRSV,
268
were obtained. As shown in Fig. 2, the plaque morphologies of BB30s and BB34s
269
were larger than that of the parental strain BB in size (Fig. 2A). Neutralization assays
270
results showed that BB10s and BB20s had a similar NA titers that were significantly
271
lower against that of BB (P
