Virus Research 204 (2015) 21–30

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Virus Research
journal homepage: www.elsevier.com/locate/virusres

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
Baochao Fan a,1 , Xing Liu a,1 , Juan Bai a , Tingjie Zhang a , Qiaoya Zhang a , Ping Jiang a,b,∗
a
Key Laboratory of Animal Diseases Diagnostic and Immunology, Ministry of Agriculture, College of Veterinary Medicine, Nanjing Agricultural University,
Nanjing 210095, China
b
Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, China

a r t i c l e i n f o a b s t r a c t

Article history: Porcine reproductive and respiratory syndrome virus (PRRSV) is mainly responsible for the heavy eco-
Received 10 January 2015 nomic losses in pig industry in the world. A number of neutralizing epitopes have been identified in the
Received in revised form 8 April 2015 viral structural proteins GP3, GP4, GP5 and M. In this study, the important amino acid (aa) residues of
Accepted 10 April 2015
HP-PRRSV strain BB affecting neutralization susceptibility of antibody were examined using resistant
Available online 20 April 2015
strains generated under neutralizing antibody (NAb) pressure in MARC-145 cells, reverse genetic tech-
nique and virus neutralization assay. HP-PRRSV strain BB was passaged under the pressure of porcine
Keywords:
NAb serum in vitro. A resistant strain BB34s with 102 and 104 aa substitutions in GP5, which have
PRRSV
102 and 104
been predicted to be the positive sites for pressure selection (Delisle et al., 2012), was cloned and
Neutralizing antibody identified. To determine the effect of the two aa residues on neutralization, eight recombinant PRRSV
strains were generated, and neutralization assay results confirmed that the aa residues 102 and 104
in GP5 played an important role in NAbs against HP-PRRSV in MARC-145 cells and porcine alveolar
macrophages. Alignment of GP5 sequences revealed that the variant aa residues at 102 and 104 were
frequent among type 2 PRRSV strains. It may be helpful for understanding the mechanism regulating
the neutralization susceptibility of PRRSV to the NAbs and monitoring the antigen variant strains in the
field.
© 2015 Elsevier B.V. All rights reserved.

1. Introduction the most economically important viruses in the swine industry

Porcine reproductive and respiratory syndrome virus (PRRSV)
is a single-stranded, positive-sense RNA virus that belongs to the
Arteriviridae family of the order Nidovirales (Gorbalenya et al.,
2006). Based on genetic and antigenic characteristics, two major
genotypes of PRRSV, type 1 (European; prototype strain Lelystad)
and type 2 (North American; prototype strain VR-2332), have been
identified and share approximately 55–70% nucleotide identity
(Andreyev et al., 1997; Mateu et al., 2006; Meng et al., 1994; Nelsen
et al., 1999). PRRSV is responsible for reproduction problems in
sows and boars, and respiratory problems in pigs of all ages (Collins
et al., 1992; Wensvoort et al., 1991). It is considered to be one of
∗ Corresponding author at: College of Veterinary Medicine, Nanjing Agricultural
University, Nanjing 210095, China. Tel.: +86 25 84395504; fax: +86 25 84396640.
E-mail address: jiangp@njau.edu.cn (P. Jiang).
1
These authors contributed equally to this work.
worldwide (Neumann et al., 2005; Pejsak et al., 1997). PRRSV
genome is a positive, single-stranded, 5 -capped and 3 -
polyadenylated mRNA molecule, with a length of approximately
15,000 nucleotides (nt). It contains, in the direction 5 –3 , two
large open reading frames (ORFs), ORF1a and 1b, which encode
the viral replicase and constitute approximately three-quarters
of the genome, and seven smaller ORFs, designated 2a, 2b and 3
through 7, which express structural proteins termed GP2a, E, GP3,
GP4, GP5, M and N, respectively (Meulenberg, 2000). An additional
structural protein, GP5a, exists and is encoded by an alternative
ORF in the subgenomic viral mRNA encoding GP5 (Johnson et al.,
2011).
PRRSV can cause persistent infections in pigs. The NAbs in
PRRSV-infected animals are generated late and their titers remain
low (reviewed by Balasuriya and MacLachlan, 2004; Lopez and
Osorio, 2004). The delayed or weak induction of NAbs upon
arterivirus infection has frequently been linked to GP5 glycosyl-
ation (Vu et al., 2011). Passive transfer of NAbs to pigs prior to

http://dx.doi.org/10.1016/j.virusres.2015.04.015
0168-1702/© 2015 Elsevier B.V. All rights reserved.
22 B. Fan et al. / Virus Research 204 (2015) 21–30
challenge with a homologous virulent PRRSV strain results in com-
plete protection of the pigs against infection, demonstrating the
important role of NAbs in protective immunity (Lopez et al., 2007;
Osorio et al., 2002). However, the role of anti-PRRSV NAbs for the
virus diversity and evolution has not been understood completely.
The genomic variation sometimes results in the emergence of
new PRRSV populations (Allende et al., 2000; Chang et al., 2002;
Rowland et al., 1999). It is expected that the adaptive immune
response of the host will act as an important source of selective
pressure in the evolutionary process of the virus (Al-Gelban, 2004;
Domingo et al., 2001). Several studies have been published on the
examination of PRRSV sequence variability followed the selective
pressure on PRRSV genes during infection in vivo. But they did not
reveal a correlation between regions under high positive selection
and the location of neutralizing B-cell epitopes (Allende et al., 2000;
Chang et al., 2002; Goldberg et al., 2003). Costers et al. (2010a,
2010b) isolated the antibody escape variants from the vaccinated
pigs and revealed that NAbs in pigs was a driving force in the rapid
evolution of the neutralizing epitope on GP4 of type 1 PRRSV strains
(Costers et al., 2010a, 2010b).
GP5 is the main target for NAbs in type 2 PRRSV strains (Gonin
et al., 1999; Ostrowski et al., 2002; Plagemann et al., 2002). A neu-
tralizing epitope (aa 37–45: SHLQLIYNL) has also been identified
on GP5 (Plagemann, 2004). A recent report argued that the major
envelope protein surface epitopes were disassociated with the virus
neutralization (Li and Murtaugh, 2012). Meanwhile, it was sug-
gested that the aa sites 102 and 104 in GP5 were likely positive
sites under some selective pressure by host immune cells based
on a large dataset of 1301 sequences (1998–2009) analysis (Delisle
et al., 2012). In this study, a type 2 highly pathogenic PRRSV (HP-
PRRSV) strain BB0907 (tenth passage, termed BB) was passaged
in MARC-145 cells in the presence of porcine serum antibodies
against this PRRSV strain, and five resistant variants were gener-
ated. The sequencing results showed that BB30s and BB34s had aa
substitutions at 102 and 104 in GP5 gene. Then eight recombinant
PRRSV strains containing these mutations were generated by site-
directed mutagenesis using BB and BB34s infectious cDNA clones.
It was found that the aa residues at 102 and 104 in GP5 of PRRSV
played important role in escaping from the NAbs against HP-PRRSV.
Our data may provide important information for understanding the
molecular mechanism regulating the neutralization susceptibility
of PRRSV to NAbs and may be helpful for monitoring the antigen
variant strains in the field.
2. Materials and methods
2.1. Cells, viruses and NAb serums
MARC-145 cells were maintained in Dulbecco’s Modified Eagle’s
medium (DMEM, GIBCO, Carlsbad, CA, USA) supplemented with
10% fetal bovine serum (FBS, GIBCO) containing 100 U penicillin/ml
and 100 ␮g streptomycin/ml at 37 ◦ C with 5% CO2 .
The HP-PRRSV isolate BB0907 (GenBank no. HQ315835, tenth
passage by using MARC-145 cells, termed BB) used in this
study was isolated in Guangxi Province, China, in 2009. The
recombinant PRRSV strains (rBB, rBB/GP5s, rBB34s, rBB34s/GP5,
rBB/GP5(Y102C), rBB/GP5(G104R), rBB/GP5s-R and rBB34s/GP5-R)
were constructed by site-directed mutagenesis of the aa residues
according to conventional methods (Fig. 1). The titers of the viral
stocks were determined by measuring their cytopathic effect (CPE)
in MARC-145 cells.
Five anti-HP-PRRSV neutralizing serums were prepared from
five 45-day-old piglets free of PRRSV by inoculated with low dose
(104 TCID50 ) of HP-PRRSV strain BB by 2 times with interval 28
days. At 70 days post-inoculation, the bloods were collected and
the serums were isolated and named N1, N2, N3, N4 and N5,
respectively. The neutralizing antibody (NA) titers of these serums
against HP-PRRSV BB were 1:34–1:48.
2.2. Isolation of antibody-resistant variants
To select variants resistant to NAb against HP-PRRSV, PRRSV
strain BB (100 TCID50 ) was mixed with a 100-fold dilution of the
anti-PRRSV antibody and incubated at 37 ◦ C for 1 h before infecting
MARC-145 cell monolayers. The virus arising from the cells show-
ing a CPE was used for the subsequent passage. After passaged by
5–30 times under the antibody pressure, five NAb-resistant vari-
ant clones were selected by using plaque tests. The titers of the
resistant virus were 106 –108 TCID50 /ml.
2.3. Sequencing of resistant variants
Viral RNA was purified from culture supernatants taken from
cells showing a CPE using the QIAmp viral RNA Mini Kit (Qia-
gen, Hilden, Germany), and reverse transcriptase PCR (RT-PCR)
was performed to generate cDNA, according to the manufacturer’s
protocol, using the SuperScript III First-Strand Synthesis Kit (Invi-
trogen, Carlsbad, CA, USA). The complete genome of the virus was
divided into seven overlapping fragments for amplification, and
the 5 and 3 termini of the genomic were synthesized using rapid
amplification of the cDNA ends (RACE). The six structural protein
genes (GP2, GP3, GP4, GP5, M and N) were amplified using the
Phanta Super Fidelity DNA Polymerase (Vazyme, China) with the
special primers.
2.4. Serum neutralization assay
The NA titers of the antibody serums against the parent and
mutant HP-PRRSVs were assayed in MARC-145 cells as described
previously (Jiang et al., 2006) with minor modifications. The
viruses were diluted to a concentration of 100 TCID50 per 50 ␮l
(103.3 TCID50 /ml) in DMEM supplemented with 2% FBS. Serial dilu-
tions of the neutralizing serums were mixed with each of the
viruses and incubated at 37 ◦ C for 1 h. The mixtures (100 ␮l/well)
were transferred to MARC-145 monolayers in 96-well plates and
incubated for an additional 4 days at 37 ◦ C with 5% CO2 . Every
serum dilution contained four replicate wells. Cells were then
examined for CPE. NA titers were expressed as the reciprocal of
the highest dilution that completely inhibited the appearance of
the CPE.
In addition, the NA titers of the antibody serums against PRRSVs
were detected in porcine alveolar macrophages (PAMs) as previ-
ously description (Vanhee et al., 2010) with minor modifications.
In brief, PAM cells were prepared from 4-week-old piglets free of
PRRSV by lung lavage and adjusted to 5 × 106 /ml with RPMI-1640
(GIBCO) medium containing 10% fetal bovine serum, 100 units/ml
of penicillin, and 100 ␮g/ml of streptomycin. The cells were added
to 96-well culture flasks (Costar, Corning Incorporated, NY) and
incubated for 6 h at 37 ◦ C in a humidified compartment to allow
cells to adhere to flasks. Twofold serial dilutions of serum N4 in
RPMI-1640 were mixed with equal volumes of 100 TCID50 PRRSV
strains, and incubated for 1 h at 37 ◦ C and transferred to a 96-well
plate (100 ␮l/well) with PAMs. The inoculum was removed after
1 h and replaced by medium, after which the cells were further
incubated for another 10 h. The cells were fixed and stained with
mAb against the N protein of PRRSV and FITC-conjugated goat anti-
mouse IgG (BOSTER, China). The NA titers were determined as the
reciprocal of the highest dilution that resulted in more than 90%
reduction of infected cells.
 B. Fan et al. / Virus Research 204 (2015) 21–30 23

Fig. 1. The construction strategy of infectious cDNA clones of the recombinant PRRSVs using BB and BF34s strains.
a
The rBB/GP5s-R and rBB34s/GP5-R were two revertant viruses and constructed from the full-length infectious cDNA clone pCMV-BB/GP5s and rBB34s/GP5 by using the
site-directed mutagenesis, respectively.

2.5. Construction of infectious cDNA clones of PRRSVs Supplementary Table S1 related to this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.virusres.2015.04.
The full-length PRRSV genome was amplified using the five 015
primer pairs listed in Table S1. A recombinant plasmid (pCMV-
BB) containing the full-length cDNA of the BB was constructed as 2.6. Rescue of recombinant viruses
shown in Fig. 1. To introduce the NAb-resistant mutations of the
structural protein GP5 into PRRSV infectious cDNA clone pCMV-BB, Plasmids carrying a full-length PRRSV cDNA were individually
site-directed mutagenesis was employed using the QuikChange® transfected into MARC-145 cells using Lipofectamine 2000 (Invitro-
II XL Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA, USA) gen) according to the manufacturer’s instructions. Four days after
according to the manufacturer’s recommendations. First, fragment transfection, the rescues of infectious viruses were obtained and
D, which encodes the structural proteins, was amplified using cloned by the plaque assay. The mutations in the rescued viruses
pCMV-BB as template and cloned into the pEASY-Simple Blunt vec- were confirmed by RT-PCR and sequencing.
tor (Beijing TransGen Biotech Co., Ltd., Beijing, China) using AscI and
SpeI restriction endonucleases (New England Biolabs, USA), thereby 2.7. Viral plaque assay
yielding pEASY-BB-D, which was used as the intermediate plasmid.
Second, the site-directed mutagenesis constructs were prepared MARC-145 cells in 12-well plates were inoculated with 100 ␮l
using pEASY-BB-D as template. Briefly, the oligonucleotide primers of tenfold serially diluted PRRSV. After 1 h adsorption at 37 ◦ C, cell
were designed such that a foreign insertion sequence was incorpo- monolayers were washed with phosphate-buffered saline (PBS)
rated at the 5 end or in the middle of the primer, leaving at least and overlaid with 1% low melting agarose in DMEM (Invitrogen)
ten nucleotides at the 3 end that matched the template sequence. containing 2% FBS. After the gel overlay solidified, the plates were
PCRs were run according to the instructions of the QuickChange
mutagenesis kit using circular plasmid DNA as the template. The
Table 1
plasmid template was eliminated by DpnI digestion (New England The recombinant viruses used in this study and the related mutant aa sites.
Biolabs), followed by transformation of the digested PCR mixtures
Name aa mutant sites Name aa mutant sites
into Top10 competent cells (Invitrogen). The intermediate plas-
mids were screened and verified by restriction enzyme mapping rBB No rBB34s No
and nucleotide sequencing. Third, the mutations in pEASY-BB-D rBB/GP5s GP5(102Y/C; 104G/R) rBB34s/GP5 GP5(102C/Y;
104R/G)
and pCMV-BB were digested with AscI/SpeI, and fragment D in rBB/GP5(Y102C) GP5(102Y/C) rBB34s/GP5-Rb No
pCMV-BB was replaced by the analogous fragments derived from rBB/GP5(G104R) GP5(104G/R)
pEASY-BB-D, and the full-length mutant clones from pCMV-BB rBB/GP5s-Ra No
were obtained. Meanwhile, the plasmid pCMV-BB34s and its site- a
The recombinant virus rBB/GP5s-R was a revertant virus from rBB/GP5s and
directed mutation plasmids were also constructed according the contained 102Y and 104G in GP5. The plasmid was constructed from the full-
above methods (Fig. 1). Besides, two different reverse mutants length infectious cDNA clone pCMV-BB/GP5s by using the site-directed mutagenesis
that were constructed from the full-length infectious cDNA clones described.
b
The recombinant virus rBB34s/GP5-R was a revertant virus from rBB34s/GP5 and
pCMV-BB/GP5s and pCMV-BB34s/GP5 by the site-directed muta- contained 102C and 104R in GP5. The plasmid was constructed from the full-length
genesis described above, respectively (Fig. 1). All the recombinant infectious cDNA clone pCMV-BB34s/GP5 by using the site-directed mutagenesis
viruses and the aa mutations are summarized in Table 1. described.
24 B. Fan et al. / Virus Research 204 (2015) 21–30

inverted (top side down) and placed into an incubator at 37 ◦ C with Table 2
Amino acid mutations in the structural proteins of the different generation viruses
5% CO2 . At 4 days post-infection (dpi), plaques were visualized by
under immune pressure.
crystal violet staining.
AA mutations

BB BB5s BB10s BB20s BB30s BB34s

2.8. Antibody binding analyses
GP2 198 G D V V V V

Parent and recombinant mutant PRRSVs were purified by ultra- GP3 69 P P S S S S

centrifugation and diluted in coating buffer to a final concentration 71K K R R R R
226 S S P P P P
5 ␮g/ml. The antigens were added in triplicate to 96-well flat-
bottomed enzyme-linked immunosorbent assay (ELISA) plates. GP4 43 D N N N N N
After incubation at 4 ◦ C for at least 16 h, wells were blocked with 44 F S S S S S

PBS-5% FBS for 1 h at room temperature. A non-saturating con- GP5 102 Y Y Y Y C C

centration of the serum antibody (diluted 1:100), which was in
the linear portion of the antibody titration curve determined pre-
viously, was added to each well, serially diluted twofold, and
incubated with the virus-coated plates for 1 h at room temperature.
After washing, anti-pig horseradish peroxidase (HRP)-conjugated
antibody was added to the plates, and they were incubated
for 1 h at room temperature. After another round of washing,
3,3 ,5,5 -tetramethylbenzidine (TMB) substrate (KPL Biomedical,
Gaithersburg, MD) was added, and 2 M H2 SO4 was used to stop
the reaction. The amount of HRP product was determined using a
plate reader at 450 nm. The control plates were coated with ultra-
centrifuged non-infected MARC-145 cell lysates.
2.9. Western blot
In order to evaluate the consistence of PRRSV antigens bind-
ing in the wells, the antigens were lysed from the coated 96-well
ELISA plate by using RIPA Lysis Buffer (Beyotime, China). Briefly,
100 ␮l lysis buffer was added into one coated well. After 5 min, the
lysate samples were obtained and added to another coated well. A
total of 12 wells (for one strain antigen) were lysed and obtained
as one sample. Then all the PRRSV strains antigen samples from
the coated plate and the original purified antigens were separated
by 12% SDS-PAGE, and transferred onto nitrocellulose filter mem-
brane (PALL, New York, USA). The membranes were incubated with
mAb N (made in our laboratory) or GP5 (provided by Dr GZ. Tong,
Shanghai Veterinary Research Institute, China) as the primary anti-
body. After the membranes were rinsed with PBS, the membrane
was treated with goat anti-mouse IgG-HRP (BOSTER, China) as the
secondary antibody. The proteins were visualized by scanning the
membranes with the Tanon 5200 chemiluminescence imaging sys-
tem (Tanon, China).
2.10. Growth curves of viruses
To determine viral one-step growth curves, PRRSV mutants
(105 TCID50 ) were inoculated into sub-confluent MARC-145 cells
or PAMs in six-well plates. Then, 200 ␮l of the supernatants of the
infected cells was collected and replenished with the same volume
of fresh medium at 12, 24, 36, 48, and 72 h post-infection (hpi), and
stored at −70 ◦ C for virus titration. The virus titers for each time
point were determined in MARC-145 cells by TCID50 .
2.11. Statistical analysis
All data were analyzed using GraphPad Prism (Version 5.03,
San Diego, California) software. Differences among all groups were
examined using one-way analysis of variance (ANOVA), followed
by Tukey’s tests. Differences between two groups were assessed
using unpaired two-tailed t-tests. Differences were considered sig-
nificant if P was <0.05.
104 G G G G R R
M 70 R R K K K K
3. Results
3.1. Generation of resistant variants against NAb to PRRSV
Five different NAb serums were prepared from the PRRSV-free
piglets infected with PRRSV strain BB. The NA titers of the serums
against this strain were 1:34–1:48. We used the N4 NAb serum that
had the highest NA titer (1:48) to generate the resistant variants
from the parental virus BB.
After 5–34 passages or purification of PRRSV BB in MARC-145
cells in the presence of N4 NAb to PRRSV, five resistant strains BB5s,
BB10s, BB20s, BB30s and BB34s, which could replicated in MARC-
145 under pressure of NAb to PRRSV, were obtained. As shown in
Fig. 2, the plaque morphologies of BB30s and BB34s were larger
than that of the parental strain BB in size (Fig. 2A). Neutralization
assays results showed that BB10s and BB20s had similar NA titers
that were significantly lower against that of BB (P < 0.05). The NA
titers of BB30s and BB34s were significantly lower than those of
BB20s (P < 0.01) and BB (P < 0.001). BB34s almost totally escaped
the neutralizing by N4 (Fig. 2B).
Meanwhile, the viral replication kinetics results showed that
BB reached peak titer at 48 hpi. BB10s and BF30s reached peak
titers at 36 and 24 hpi, respectively. BB34s had the highest titers
(109 TCID50 /ml) in these six strains (Fig. 2C). It suggested that BB34s
could replicate more efficiently in MARC-145 than the parental
virus BB.
Furthermore, the replication of those resistant strains viruses
in PAMs was detected with or without NAb to PRRSV. The results
showed that the NA titers of N4 serum with BB30s and BB34s
were significantly lower than those with their parental virus BB
(P < 0.001) (Fig. 2D). It indicated that they escaped efficiently from
the neutralizing by N4. But they could replicate efficiently in PAMs
as BB (Fig. 2E).
3.2. Sequence analyses of the NAb resistant variants
To map the binding epitope defined by the NAb serum, the struc-
tural protein genes of the resistant strains and parent strain BB were
sequenced and analyzed. As shown in Table 2, BB34s had a total of
nine different aa substitutions in the GP2 (G198V), GP3 (P69S, K71R,
and S226P), GP4 (D43N and F44S), GP5 (Y102C and G104R) and M
(R70K) proteins, compared with the parent PRRSV BB. Meanwhile,
BB34s had only different aa residues at 102 and 104 in GP5, when
compared with BB10s and BB20s.
3.3. Identification of the antibody binding sites
To elucidate the role of the two aa mutations at 102 and 104
in GP5 of BB34s in the virus’s escape ability, four recombinant
 B. Fan et al. / Virus Research 204 (2015) 21–30 25

Fig. 2. Generation and characterization of the resistant variants from PRRSV BB under NAb to HP-PRRSV in MARC-145 cells (A–C) and PAMs (D and E). The neutralization
assays of the variants were generated under N4 neutralizing serum in MARC-145 cells. The neutralization titer represents the inverse of the highest antibody dilution at
which an infectivity of 100 TCID50 of the viruses was neutralized. (A) Analysis of plaque morphologies. (B and D) Virus neutralization assays. (C and E) The growth kinetics.
The data are represented as the means ± s.d. of three independent experiments, and significant differences are shown (*P < 0.05 and ***P < 0.001) by using one-way analysis
of variance (ANOVA).

PRRSV strains, rBB, rBB/GP5s, rBB34s and rBB34s/GP5 were res-
cued by mutations of both aa residues at 102 and 104 by using
reverse genetic techniques as Fig. 1. In addition, in order to definite
that the results were due to the aa mutants, two revertant viruses
rBB/GP5s-R and rBB34s/GP5-R were also generated.
Plaque assay results showed that the plaques of rBB/GP5s were
larger than those of rBB, and the plaques of rBB34s/GP5 were
smaller than those of rBB34s (Fig. 3A). The NA titer of the N4
serum with rBB/GP5s was significantly lower than that with rBB
(P < 0.001). The NA titers of the same serum with rBB34s/GP5 were
significantly higher than that with rBB34s (P < 0.001). Meanwhile,
the revertant viruses rBB/GP5s-R and rBB34s/GP5-R had the simi-
lar NA titers with rBB and rBB34s, respectively (Fig. 3B). These data
suggested that the two aa residues at 102 and 104 in GP5 were crit-
ical for binding the NAb. Moreover, viral replication kinetic results
showed that rBB/GP5s and rBB34s had a higher replication capacity
than rBB and rBB34s/GP5 (Fig. 3C).
3.4. Binding analysis of resistant variants
ELISA binding was used to confirm that the resistant ability of
the viruses was due to the antigenic mutations. As shown in Fig. 4A,
the binding antigens in the plates that coated with the purified
viruses at the same concentration almost had the same amount
of GP5 and N proteins. Meanwhile, the ELISA results showed that
the evasion virus BB34s demonstrated significantly lower bind-
ing ability to N4 serum compared with its parental virus BB. The
rBB/GP5s had higher binding inability to N4 than did rBB. rBB34s
and its parent virus BB34s had the lowest OD values compared with
other virus groups. However, the aa mutant viruses rBB34s/GP5
26 B. Fan et al. / Virus Research 204 (2015) 21–30

Fig. 3. Characterization of the recombinant PRRSV strains rBB, rBB/GP5s, rBB34s, rBB34s/GP5 and two revertant viruses rBB/GP5s-R and rBB34s/GP5-R. (A) Plaque morphology
assay. (B) Virus neutralization assays of recombinant viruses by using the N4 serum. (C) The growth kinetics. The data are represented as the means ± s.d. of three independent
experiments, and significant differences are shown (***P < 0.001) by using one-way analysis of variance (ANOVA).

significantly increased the N4 binding ability and had similar OD
values to BB (Fig. 4B).
3.5. Identification of aa mutations Y102C and G104R in GP5
In order to determine the key aa site in GP5 played greater
role in the neutralization abilities, we constructed another two
recombinant viruses that contained only one aa mutation Y102C
or G104R in GP5 using plasmid pCMV-BB (Fig. 1). As shown in
Fig. 5A, the recombinant viruses rBF10/sGP5(Y102C) formed larger
plaques than rBB/GP5s(G104R) and rBB. The neutralization abili-
ties of rBB/GP5s(Y102C) and rBB/GP5s(G104R) were both obviously
decreased when compared with that of rBB (P < 0.05) (Fig. 5B).
Meanwhile, rBB/GP5s(Y102C) had a higher replication capacity
than rBB/GP5s(G104R) and rBB (Fig. 5C).
rBB34s/GP5 were significantly higher than those with BB34s. It con-
firmed that the aa residues 102 and 104 in GP5 played important
role in the binding ability of PRRSV to its NAb.
3.7. Replication of the recombinant PRRSV in PAMs
The NA titers of N4 serums antibody were measured with the
different mutations in PAMs. As shown in Fig. 6A, rBB34s signif-
icantly reduced the NA titers of the serum when compared with
rBB (P < 0.001). Meanwhile, the NA titer with rBB/GP5s was signif-
icantly lower than that with rBB (P < 0.01). And the NA titers of the
same serum with rBB34s/GP5 were significantly higher than that
with rBB34s (P < 0.01). However, the viral replication kinetics of the
four virus strains rBB, rBB/GP5s, rBB34s, rBB34s/GP5 were similar
to each other (Fig. 6B).

3.6. Resistances of the variants against different NAb serums to
HP-PRRSV
The NA titers of five serums antibody samples were mea-
sured with the different strains obtained as above. As shown
in Table 3, the NA titers with BB34s and rBB/GP5s were
significantly reduced when compared to those with the parent
virus BB. Meanwhile, the neutralizing abilities of the serums with
Table 3
The neutralizing antibody titers of the five serums against the four different
PRRSV strains. The data are represented as the means ± s.d. of three independent
experiments.
3.8. Comparison of the aa sequences of PRRSV isolates in the field
As shown in Fig. 7, some field isolates also had the same aa
mutants as BB34s and BB30s. For example, strains HUBEZ1 and
JMS01 possessed the Y102C substitution, while HN10 possessed
the G104R substitution in GP5, as did BB34s. PRRSV strains GD and
SZ1-09 had some other aa substitutions at these two sites (Y102L
and G104E). It was also noticed that aa 102 of the classical PRRSV
strains VR2332 and S1 was valine (V), which is different from those
of HP-PRRSV strains, such as SY0608 and JXA1.
4. Discussion

Virus Neutralizing antibody titers (mean ± s.d.)

PRRSV has been proved to be highly genetically variable (Leng
N1 N2 N3 N4 N5 et al., 2014), and a number of antigenic sites recognized by NAbs
BB 38 ± 5.9 34 ± 3.8 42 ± 4.8 48 ± 6.7 40 ± 2.8 have been identified in the viral structural proteins (Meulenberg
BB34s 3 ± 2.9 2 ± 1.9 4 ± 3.6 4 ± 2.2 3 ± 0.9 et al., 1997; Oleksiewicz et al., 2001, 2005; Plagemann, 2004).
rBB/GP5s 8 ± 2.5 6 ± 2.9 8 ± 3.3 10 ± 3.3 6 ± 1.6 However, the key aa residues related to viral neutralization sus-
rBB34s/GP5 31 ± 3.2 28 ± 5.3 34 ± 4.3 34 ± 5.2 31 ± 1.8
ceptibility to the porcine serum neutralizing antibody have not
 B. Fan et al. / Virus Research 204 (2015) 21–30 27

Fig. 4. Binding analysis of the escape and recombinant mutant strains. (A) The purified antigens and binding antigens of PRRSV strains from the coated 96-well ELISA plate
were detected by Western blot with the monoclonal antibodies against GP5 and N proteins of PRRSV. (B) A binding ELISA was performed using plates coated with each of the
indicated viruses in triplicate. Serial dilutions of N4 serum were added to the wells, followed by the addition of a secondary anti-pig antibody conjugated to HRP. OD values
were read at 450 nm. The negative control was wells that coated with ultracentrifuged non-infected MARC-145 cell lysates.

been understood completely. In this study, the HP-PRRSV BB strain
was passaged in MARC-145 cells under the pressure of a serum
NAb against HP-PRRSV. And five resistant variants that signifi-
cantly reduced the NA titers compared with BB were generated.
The sequencing results showed that the BB34s and BB30s contained
two aa mutations Y102C and G104R in GP5 when compared to
BB10s and BB20s, which being suggested as positive sites for evo-
lutionary selection by analyzed a large dataset of 1301 sequences
(1998–2009) (Delisle et al., 2012). The consistency of the aa sites
promoted us to investigate the importance of these two sites. By
using the reverse genetic techniques and the neutralization assay,
it was firstly confirmed that the two aa residues, 102 and 104 within
GP5 of PRRSV played important role in escaping from NAbs against
PRRSV.
The major structural proteins, GP5 and M, form disulfide-linked
heterodimers that bind to heparin sulfate, which is required for
virus attachment (Delputte et al., 2005; Faaberg et al., 1995; Snijder
et al., 2003). Many reports implicate GP5 and M as key PRRSV neu-
tralization targets (Ostrowski et al., 2002; Pirzadeh and Dea, 1997;
Plagemann, 2004; Plagemann et al., 2002; Weiland et al., 1999;
Wissink et al., 2003; Yang et al., 2000). The specific sequences of
linear neutralizing epitopes in GP5 have been identified as aa 37–45
(SHLQLIYNL) and aa 36–52 (SSHLQLIYNLTLCELNG) of the North
American strain VR-2332 (Ostrowski et al., 2002; Plagemann et al.,
2002). Synthetic peptides representing neutralizing epitopes from
PRRSV GP5 did not induce virus neutralizing antibodies, suggesting
that additional residues or structural features are critical for anti-
genicity (Lopez and Osorio, 2004; Plagemann, 2001). In this study,
we identified two aa sites, 102 and 104, which were the important
antibody binding sites in GP5 using an immune pressure system in
vitro. A TMHMM prediction shows that this region will be predicted
to locate in one putative, short ectodomain region (aa 100–109)
after two transmembrane domains prior to the third transmem-
brane region in GP5 (Li and Murtaugh, 2012; Mardassi et al., 1995,
1996). Considering the important role of the two aa substitutions
(102 and 104) in preventing neutralization by antibodies, as well as
the fact that no other aa substitutions occurred in GP5, especially
in the confirmed neutralizing epitopes, it is likely that the two aa
substitutions may be one part for formation of the conformational
neutralizing epitopes in GP5. Of course, this hypothesis should be
28 B. Fan et al. / Virus Research 204 (2015) 21–30

Fig. 5. Characterization of the recombinant PRRSV rBB/GP5(Y102C) and rBB/GP5(G104R). (A) Plaque morphology assays. (B) Virus neutralization assays of recombinant
viruses by using N4 neutralizing serum. (C) The growth kinetics. The data are represented as the means ± s.d. of three independent experiments, and significant differences
are shown (*P < 0.05, **P < 0.01 and ***P < 0.001) by using one-way analysis of variance (ANOVA).

investigated further after the three-dimensional model of PRRSV
GP5 obtained.
The in vitro generation of escape mutants is unlikely to recapitu-
late drift in its entirety. This is largely due to the stochastic nature of
drift and the very large mutational space available. The exertion of
antibody-mediated selective pressure onto a neutralizing epitope
rapidly resulted in the selection of viable type 1 PRRSV variants that
were resistant to neutralization by these antibodies. It was found
that NAbs in pigs might be a driving force in the rapid evolution
of the neutralizing epitope on GP4 of type 1 PRRSV strains (Costers
et al., 2010a, 2010b). In this study, the sequence results showed
that the structure proteins GP2, GP3, GP4 and M of the resistant
strains also contained some aa substitutions compared with the
parent viruses. In addition, the complete genome sequence analy-
sis results of BB34s (GenBank no. KM453698) revealed that some aa
mutants also occurred in the non-structural proteins (Nsps) of this
strain when compared to BB (data not shown). The resistant variant
BB34s replicated more efficiently in MARC-145 than the parental

Fig. 6. Characterization of the recombinant PRRSV strains in PAMs. (A) Virus neutralization assays of recombinant viruses by using N4 neutralizing serum. (B) The growth
kinetics of recombinant viruses. The data are represented as the means ± s.d. of three independent experiments, and significant differences are shown (**P < 0.01 and
***P < 0.001) by using one-way analysis of variance (ANOVA).
 B. Fan et al. / Virus Research 204 (2015) 21–30
Fig. 7. Alignment of partial GP5 aa sequences of BB34s with wild-type PRRSV isolates. Y102C and G104R in GP5 of BB34s and the same sites of the field PRRSV isolates are
indicated by black boxes.
virus BB. The changes in Nsps of BB34s upon long term passaging
in a host cell might suggest further adaptation of the virus to the
host cell machinery.
Using monoclonal antibodies (mAbs) directed against a sepa-
rate protein, especially the mAbs against conformational epitopes,
may lead to the comprehensive identification of neutralizing epi-
topes. The antigenic sites of influenza virus have been identified
using immune pressure method with the specific mAbs (Hensley
et al., 2009; O’Donnell et al., 2012). In this study, we identified the
antigenic sites using an anti-PRRSV neutralizing polyclonal serum
made in swine. In addition, during the passage of PRRSV strain BB,
we used one negative anti-PRRSV serum as negative pressure con-
trol. The sequence results indicated that the viruses at passage 10
and 34 do not have aa substitutions at aa102 and aa104 in GP5
(data not shown here). Of course, further studies should be needed
to improve these techniques and to address the importance of other
putative antigenic sites using different anti-PRRSV serums.
In addition, we noted that the significantly resistant virus BB30s
was generated on the passage 30. It takes a long time when com-
pared with the mAb-resistant PRRSV strains of LV generated on
passage 5 (Costers et al., 2010a). The possible reasons for this dif-
ference might be related to the techniques of cloning and passaging
and the NAb serum. The mixed clones might be picked up during
the passage of those virus clones. At passage 34s, there were still
virus particles present that were not resistant yet. The putative aa
residues in other proteins might regulate the viral neutralization
susceptibility to NAb, which should be investigated in the further.
In conclusion, by passage of PRRSV under the viral specific NAb
pressure, it was firstly found that the aa residues 102 and 104 in
GP5 of PRRSV played important role in regulating neutralization
susceptibility to the porcine serum NAb. It may be helpful for mon-
itoring the antigen variant strains in the field and developing new
vaccine against PRRSV in the future.
29
Acknowledgments
This work was mainly supported by the National Natural Science
Foundation (31230071), grants from the Ministry of Education,
China (313031, 2012009711004) for PRRSV immunology, a grant
from the Ministry of Agriculture (CARS-36) for swine disease
controlling techniques, and the priority academic program devel-
opment of Jiangsu higher education institutions (PAPD).
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