Bài tập tổng hợp - C4

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O R I G I N A L A R T I C L E

Sequence and Phylogenetic Analyses of the Nsp2 and ORF5

Genes of Porcine Reproductive and Respiratory Syndrome

Virus in Boars from South China in 2015

P. P. Wang1,*, J. G. Dong1,2,*, L. Y. Zhang1, P. S. Liang1, Y. L. Liu1, L. Wang1, F. H. Fan3and C. X. Song1

1_{College of Animal Science & National Engineering Center for Swine Breeding Industry, South China Agriculture University, Guangzhou, China}
2_{Xinyang Animal Disease Prevention and Control Engineering Research Center, Xinyang College of Agriculture and Forestry, Xinyang, China}
3_{Testing Center of Breeding Swine Quality of China Ministry of Agriculture, Guangzhou, China}

Keywords:

Porcine reproductive and respiratory
syndrome virus; Nsp2; GP5; mutation;
phylogenetic analysis

Correspondence:

C. X. Song. College of Animal Science &
National Engineering Center for Swine
Breeding Industry, South China Agriculture
University, Guangzhou 510642, China.
Tel.: +86 13829723528;

E-mails: ;

and

F. H. Fan. Testing Center of Breeding Swine
Quality of China Ministry of Agriculture,
Guangzhou 510500, China.

Fax: 02087038106;
E-mail:

*These authors contributed equally to this
work.

Received for publication September 8, 2016
doi:10.1111/tbed.12594

Summary

Porcine reproductive and respiratory syndrome virus (PRRSV) is highly
geneti-cally diverse; however, little is known about the molecular epidemiology of
PRRSV in the boar farms of South China. In this study, 367 samples were
col-lected from boar farms in South China in 2015. The Nsp2 hypervariable region
and ORF5 gene were PCR amplified from 66 PRRSV-positive samples, followed
by sequencing and analysis. The percentage of PRRSV antigen-positive samples
was 17.98%; 8.72% were positive for highly pathogenic PRRSV (HP-PRRSV), and
9.26% were positive for low pathogenic PRRSV (LP-PRRSV). Sequence alignment
and phylogenetic tree analyses revealed three novel patterns of deletion in the
hypervariable region of Nsp2, which had not been identified previously.
Further-more, numerous amino acid substitutions were identified in the putative signal
peptide and extravirion regions of GP5. These results demonstrate for the first
time that the existence of multiple different strains on the same boar farm, and
extensive genetic mutation and high infection rate of PRRSV in boars from South
China. Our research contributes to the understanding of the epidemiology and

genetic characteristics of PRRSV on boar farms.

Introduction

Porcine reproductive and respiratory syndrome (PRRS) is
an economically important infectious disease of swine and
is characterized by reproductive failure and respiratory
dis-ease both in sows and in pigs of all ages respectively
(Albina, 1997; Pejsak et al., 1997). The disease was first
reported in the USA in the late 1980s, and over the
succeed-ing years, has become one of the most important diseases
in the swine industry, being identified in pigs worldwide
(Albina et al., 1992).

The causative agent of this disease, PRRS virus (PRRSV),
was first identified in 1991 in the Netherlands, and then in
the USA in 1992 (Keffaber, 1989; Wensvoort et al., 1991).
Porcine reproductive and respiratory syndrome virus is an
enveloped, single-stranded, positive-sense RNA virus,
which is classified into the order Nidovirales, family

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autoproteolytically cleaved into fourteen non-structural
proteins (Nsps): Nsp1a, Nsp1b, Nsp2/3/4/5/6, Nsp7a,
Nsp7band Nsp8/9/10/11/12 (van Dinten et al., 2000;
Bau-tista et al., 2002; Beerens et al., 2007). Open reading frame
2a, ORF2b and ORFs3/4/5/6/7 encode structural proteins,
including GP2, E, GP3, GP4, GP5, GP5a, M and N
(Bau-tista et al., 1996; Johnson et al., 2011).

Porcine reproductive and respiratory syndrome virus

can be classified into two genotypes: type 1 PRRSV
(Euro-pean genotype) is represented by the prototype strain
Lelys-tad virus, while type 2 PRRSV (North American genotype)
is represented by the prototype strain VR-2332. The two
types of PRRSV share only approximately 60% nucleotide
identity; however, they both cause similar syndromes in
pigs (Forsberg, 2005). Among all the PRRSV genes, those
encoding Nsp2 and GP5 are highly variable and are often
used for phylogenetic analyses and molecular epidemiology
research (Murtaugh et al., 1995; Cha et al., 2004).

In China, an outbreak of PRRS was documented in
1995, following which the disease spread to almost all
provinces, with serious effects on the Chinese swine
industry (Zhou and Yang, 2010). In 2006 in particular,
there was an outbreak of a porcine syndrome
character-ized by prolonged high-fever, anorexia, rubefaction, high
morbidity and mortality, in south China, which quickly
spread throughout the country, leading to unprecedented
economic losses for the Chinese pork industry (Li et al.,
2007). Further investigation indicated that the pathogen
causing the porcine high-fever syndrome was highly
pathogenic PRRS virus (HP-PRRSV) (Li et al., 2007).
From 2006 until now, many studies of the molecular
epidemiology of PRRSV have been reported; however,
there have been no studies of the epidemiology of
PRRSV in infected boars.

To fully understand the molecular epidemiology for
PRRSV in boars in South China, we collected blood

sam-ples from boars in different provinces and sequenced the
PRRSV Nsp2 hypervariable (HV) region and ORF5 genes
of positive samples. In addition, the evolutionary
character-istics of Chinese PRRSV were analysed.

Materials and Methods

Sample collection and antibody detection

A total of 367 blood samples from healthy boars (breeds:
Duroc, Large White and Landrace) of approximately 25 kg
body weight from 31 pig farms located in Guangdong,
Fujian, Zhejiang and Guangxi provinces of South China
were submitted to our laboratory between 21 August 2015
and 13 October 2015. The mean volume of each blood
sample was 10 ml. These samples were used for
amplifica-tion and sequencing of the Nsp2 HV region and ORF5
genes.

RNA extraction and RT-PCR

Total RNA was extracted from blood samples using TRIzol
reagent (Life Technologies, New York, NY, USA) according
to the manufacturer’s instructions. Reverse transcription
was performed in a total volume of 20 ll containing
10.5ll total RNA, 4 ll 59 reverse transcription buffer,
2ll deoxynucleoside triphosphate (dNTP) mixture
(10 mM), 1 ll 9-mer random primers (50 pM), 2ll reverse

transcriptase (5 U/ll; M-MLV, Takara) and 0.5 ll RNase

inhibitor (40 U/ll). The reactants were mixed gently,
placed in a water bath at 42°C for 1 h, then incubated on
ice for 2 min. Primers to amplify the Nsp2 HV region were:
forward primer, 50-CTCCGTGGTGCAACAA-30; reverse
primer, 50-GGCTTGAGCTGAGTAT-30; The primers to
amplify the ORF5 gene were: forward primer, 50
-ATGTTGGGGAAGTGCTT-30; reverse primer, 50
-GAC-GACCCCATTGTT-30. Amplification was performed using
the following reaction conditions: one cycle at 94°C for
5 min; 30 cycles at 94°C for 30 s, 57°C for 45 s and 72°C
for 30 s, followed by incubation at 72°C for 10 min, and a
final hold at 4°C. PCR products were visualized by 1%
agarose gel electrophoresis and ultraviolet light.

Cloning and nucleotide sequencing

The PCR products were purified using the Wizard SV Gel
and PCR Clean-Up system (Promega, Madison, WI, USA),
and then cloned into the pMD18-T vector (TaKaRa
Biotechnology Co., Ltd., Dalian, China). Plasmids were
submitted to BGI (Beijing, China) for sequencing.

Sequence alignment and phylogenetic analysis

Nucleotide and deduced amino acid (AA) sequences were
aligned using the MegAlign program of DNASTAR7.0
soft-ware (DNASTAR, Inc., Madison, WI, USA) to determine
sequence homology. A phylogenetic tree was constructed
using MEGA5.2 software (www.megasoftware.net/. Tempe,
AZ, USA) with the neighbour-joining method; bootstrap

values were calculated for 1000 replicates for alignment
with multiple sequences of representative PRRSV sequences
available in GenBank (Table 1).

Results

Epidemiology of PRRSV in South China

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GenBank accession numbers for the Nsp2 HV genes
were KX010603–KX010668, and for the ORF5 genes
were KX000300–KU997091 (Table 2). The percentage
positive for PRRSV antigen by PCR were calculated
(Table 3). In total, 17.98% (66) samples were positive
for PRRSV antigen. In addition, 8.72% (32/367) were
positive for HP-PRRSV antigen, and 9.26% (34/367)
were positive for the low pathogenic PRRSV
(LP-PRRSV) antigen. In Guangdong, percentages of samples

positive for PRRSV antigen was 21.57 (66/306)
respec-tively (Fig 1a), of which, 10.46 (32/306) and 11.11 (34/
306) were positive for HP-PRRSV and LP-PRRSV
anti-gens respectively. All samples from Fujian, Zhejiang and
Guangxi provinces were negative for PRRSV antigen
(Fig. 1d). The PRRSV antigen-positive percentages in
samples from different areas of Guangdong were
anal-ysed further, demonstrating that there were higher rates
of PRRSV antigen infection in the Pearl River Delta
Table 1. Information of the representative strains

Strain Year Area Accession No. Strain Year Area Accession No.

EDRD-1 Japan 1992 AB288356 JX143 China 2008 EU708726

ATCC VR-2332 America 1993 U87392 08HuN China 2008 GU169411

Leystad virus Netherlands 1993 M96262 CWZ-1-F3 China 2008 FJ889130

BJ-4 Beijing, China 1996 AF331831 GDBY1 China 2008 GQ374442

CH-1a Beijing, China 1996 AY032626 NT0801 China 2008 HQ315836

PL97-1 Korea 1997 AY585241 YN2008 China 2008 EU880435

16244B America 1998 AF046869 YN9 China 2008 GU232738

MLV Resp PRRS/Repro America 1999 AF159149 NADC30 America 2008 JN654459

SP Singapore 1999 AF184212 CA-2 Korea 2008 KF555450

EuroPRRSV America 1999 AY366525 TP P90 China 2009 GU232737

NVSL 97-7985 IA 1-4-2 America 2000 AF325691 GD-100 China 2009 GU143913

PA8 Canada 2000 AF176348 09HEB China 2009 JF268679

Jam2 Japan 2000 AB811787 09HEN1 China 2009 JF268684

HB-1(sh)/2002 Hebei, China 2002 AY150312 09HUB1 China 2009 JF268682

HB-2(sh)/2002 Hebei, China 2002 AY262352 SD0901 China 2009 JN256115

P129 America 2002 AF494042 SX2009 China 2009 FJ895329

HN1 Henan, China 2003 AY457635 DC China 2010 JF748718

GS2003 China 2003 EU880442 FS China 2010 JF796180

VR-2332 America 2003 AY150564 GX1003 China 2010 JX912249

JA142 America 2003 AY424271 QY2010 China 2010 JQ743666

NB/04 Zhejiang, China 2004 FJ536165 Yamagata10-7 Japan 2010 AB811788

Resp PRRS MLV America 2005 AF066183 Aomori10-5 Japan 2010 AB811789

SHB Guangzhou, China 2005 EU864232 DY China 2011 JN864948

MN184A America 2005 DQ176019 GM2 China 2011 JN662424

01NP1.2 Thailand 2005 DQ056373 HH08 China 2011 JX679179

Ingelvac ATP America 2006 DQ988080 QYYZ China 2011 JQ308798

JXwn06 China 2006 EF641008 Nagasaki11-14 Japan 2011 AB811786

JXA1 China 2006 EF112445 10-10JL China 2012 JQ663554

TJ China 2006 EU860248 JL-04/12 China 2012 JX177644

GD China 2006 EU825724 SD16 China 2012 JX087437

TP China 2006 EU864233 JL580 China 2013 KR706343

Jsyc China 2006 EU939312 DK-1997-19407B Denmark 2013 KC862576

HB-1/3.9 China 2007 EU360130 DK-2012-01-11-3 Denmark 2013 KC862575

HuN4 China 2007 EF635006 KNU-12-KJ4 Korea 2013 KF555451

Em2007 China 2007 EU262603 HP/Thailand/19500LL/2010 Thailand 2013 KF735060

BJsy06 China 2007 EU097707 NMG2014 China 2014 KM000066

Henan-1 China 2007 EU200962 HB2014001 China 2014 KM261784

SY0608 China 2007 EU144079 NVDC-SC1-2014 China 2014 KP771739

WUH1 China 2007 EU187484 NVDC-13SXJC-2014 China 2014 KP771780

Shaanxi-2 China 2007 HQ401282 BB0907-F44 China 2014 KM453699

CG China 2007 EU864231 HENAN-HEB China 2014 KJ143621

CH-1R China 2008 EU807840 CHsx1401 China 2015 KP861625

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(central Guangdong Province) (26.18%) and north
Guangdong (28.00%), compared with east (13.51%) and
west (7.55%) Guangdong (Fig. 1d).

Alignment and analysis of Nsp2 sequences

To explore the sequence characteristics of PRRSV in boars,
DNA fragments from the Nsp2 HV region of 66 positive
samples were amplified and sequenced. The results
demon-strated that all of the 66 amplified Nsp2 HV regions shared
73.0–76.1%, 83.5–90.5% and 87.6–97.5% nucleotide
iden-tity with the representative strains VR-2332 (American),
CH-1a (Chinese LP-PRRSV) and JXA1 (Chinese
HP-PRRSV) respectively. Nucleotide identity with the
Euro-pean genotypic representative strain Lelystad virus (LV)
was only 41.8–44.6%. These results indicate that the
detected strains belonged to the North American genotype
group. Comparisons of the deduced Nsp2 AA sequences
from the 66 positive samples revealed extensive mutations
in the Nsp2 HV region (Fig. 2). Notably, three different
types of deletions were observed in the Nsp2 HV region
(Fig. 2). Compared with VR-2332 and CH-1a strains, 30
positive samples had deletions of 30 AAs (AAs 482 and
533–561), which is a characteristic of HP-PRRSV (Zhou
et al., 2008). Among the other 36 positive samples, 30 had
an 8-AA deletion (AA478–485), which is different from the
reference strains. Two samples, GDSZF1-1 and -3, had a
20-AA deletion (AA533–552), whereas four samples
(GDQYF1-3,-4,-5 and -7) had no deletions, and were
highly similar to CH-1a. No NADC30-like Nsp2
sequence-containing strains were found among the isolates in this
study (Zhao et al., 2015; Zhou et al., 2015).

Phylogenetic analysis using Nsp2 gene sequences

To understand the genetic relationships among the 66
PRRSV isolates from this study and other representative
strains, phylogenetic trees were constructed using the
neighbour-joining method, based on the Nsp2 HV region
AA sequences. As shown in Fig. 3, the 66 isolates from this
study and the reference strains could be divided into three
subgenotypes; the representative North American and
Southeast Asian strains were classified into subgenotype I.
None of the strains identified in this study were closely
related to subgenotype I. Thirty-four isolates clustered into
subgenotype II, along with the representative strains CH-1a
and SHB. The other 32 positive samples formed a large
cluster in subgenotype III. Subgenotype II could be further
divided into two groups, with isolates GDQYF1-3 and -4
belonging to group I, and the other 32 isolates to group II,
and sharing identity with the SHB strain. Subgenotype III
was divided into four groups: GDSZF1-1 and -3 belonged
to group II and shared identity with the strains HB-1-3.9
and HB-1 sh 2002. GDGZF1-8, 2–18 and -19, GDJMF1-1
and GDMZF3-2 belonged to group III and shared identity
with TP-P90; GDJMF1-2 and GDZQF3-1 and -3 belonged
to group IV and shared identity with JXA1-P80 and
GX1003, and the other 21 isolates belonged to group I.
Phylogenetic analysis using ORF5 gene sequences

To further investigate the genetic relationships of the 66
PRRSV isolates with other representative strains,
phyloge-netic trees were constructed using the neighbour-joining
method, based on the AA sequences encoded by ORF5 of

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the isolates from this study and representative PRRSV
sequences previously deposited in the database. The results
showed that the 66 isolates from this study and the
refer-ence strains could be divided into four subgenotypes
(Fig 4). The NADC30-like strains CHsx1401, JL580 and
HENAN-HEB (which are new mutated strains, isolated in
recent years) (Zhao et al., 2015; Zhou et al., 2015)
belonged to subgenotype I. The LP-PRRSV representative
strain, CH-1a, and its cell-attenuated live vaccine strain,
CH-1R, belonged to subgenotype II, which also included
the North American type representative strain VR2332.
None of the strains isolated in this study were closely
related to subgenotypes I or II. GDQYF1-5 and -6 belonged
to subgenotype III, which contained the representative
strains SHB and HB-1-3.9. The other isolates from this
study belonged to subgenotype IV, which also contained
the highly pathogenic strains JXA1, JXwn06 and HuN4.

Subgenotype IV could be further divided into three groups,
of which 24 positive samples belonged to group I and
shared identity with the recently isolated highly pathogenic
strain NMG2014 and the cell-attenuated strain GD-100,
while eight other isolates from this study were closely
related to one another and belonged to group II, which
contained the representative strain JXA1 and its
cell-attenu-ated live vaccine strain, JXA1-P80. Finally, the remaining
three strains, GDQYF1-1, -2 and -7 belonged to group III,
and shared high identity with the cell-attenuated live
vac-cine strain, TP-P90.

Sequence alignment and analysis of GP5

The GP5 nucleotide and AA sequences of all 66 positive
samples were of the same size and had no nucleotide or AA
deletions or insertions, when compared with representative
Table 2. Geographic origin and amplified sequence size from clinical samples in this study

No. Designation Area ORF5 (bp) Nsp2 (bp) No. Designation Area ORF5 (bp) Nsp2 (bp)

1 GDGZF1-1 Guangzhou 603 1035 34 GDGZF2-20 Guangzhou 603 969

2 GDGZF1-2 Guangzhou 603 1035 35 GDJMF1-1 Jiangmen 603 969

3 GDGZF1-3 Guangzhou 603 1035 36 GDJMF1-2 Jiangmen 603 969

4 GDGZF1-4 Guangzhou 603 1035 37 GDJMF3-1 Jiangmen 603 1035

5 GDGZF1-5 Guangzhou 603 1035 38 GDJMF3-2 Jiangmen 603 969

6 GDGZF1-6 Guangzhou 603 1035 39 GDMZF3-1 Maoming 603 1035

7 GDGZF1-7 Guangzhou 603 969 40 GDMZF3-2 Maoming 603 969

8 GDGZF1-8 Guangzhou 603 969 41 GDMZF3-3 Maoming 603 1035

9 GDGZF1-9 Guangzhou 603 969 42 GDHYF2-1 Heyuan 603 969

10 GDGZF1-10 Guangzhou 603 1035 43 GDHYF2-2 Heyuan 603 969

11 GDGZF1-11 Guangzhou 603 1035 44 GDZQF3-1 Zhaoqing 603 969

12 GDGZF1-12 Guangzhou 603 1035 45 GDZQF3-2 Zhaoqing 603 1035

13 GDGZF1-13 Guangzhou 603 1035 46 GDZQF3-3 Zhaoqing 603 969

14 GDGZF1-14 Guangzhou 603 1035 47 GDZQF3-4 Zhaoqing 603 1035

15 GDGZF2-1 Guangzhou 603 969 48 GDZQF3-5 Zhaoqing 603 1035

16 GDGZF2-2 Guangzhou 603 969 49 GDQYF1-1 Qingyuan 603 1035

17 GDGZF2-3 Guangzhou 603 969 50 GDQYF1-2 Qingyuan 603 1035

18 GDGZF2-4 Guangzhou 603 1035 51 GDQYF1-3 Qingyuan 603 1059

19 GDGZF2-5 Guangzhou 603 969 52 GDQYF1-4 Qingyuan 603 1059

20 GDGZF2-6 Guangzhou 603 1035 53 GDQYF1-5 Qingyuan 603 1059

21 GDGZF2-7 Guangzhou 603 1035 54 GDQYF1-6 Qingyuan 603 969

22 GDGZF2-8 Guangzhou 603 969 55 GDQYF1-7 Qingyuan 603 1059

23 GDGZF2-9 Guangzhou 603 1035 56 GDZJF1-1 Zhanjiang 603 1035

24 GDGZF2-10 Guangzhou 603 1035 57 GDZJF1-2 Zhanjiang 603 969

25 GDGZF2-11 Guangzhou 603 969 58 GDZJF1-3 Zhanjiang 603 969

26 GDGZF2-12 Guangzhou 603 969 59 GDZJF1-4 Zhanjiang 603 969

27 GDGZF2-13 Guangzhou 603 969 60 GDSZF1-1 Shenzhen 603 1002

28 GDGZF2-14 Guangzhou 603 969 61 GDSZF1-2 Shenzhen 603 1035

29 GDGZF2-15 Guangzhou 603 1035 62 GDSZF1-3 Shenzhen 603 1002

30 GDGZF2-16 Guangzhou 603 1035 63 GDSZF1-4 Shenzhen 603 1035

31 GDGZF2-17 Guangzhou 603 969 64 GDSZF1-5 Shenzhen 603 969

32 GDGZF2-18 Guangzhou 603 969 65 GDSZF1-6 Shenzhen 603 969

33 GDGZF2-19 Guangzhou 603 969 66 GDSZF1-7 Shenzhen 603 1035

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strains. Sequence alignments indicated that AA sequence
identities among the 66 isolates ranged from 98.2–99.2%,
with 96.5–97.6%, 97.6–99.2% and 98.9–99.2% AA
similar-ity among strains VR-2332, CH-1a and JXA1, respectively.
Compared with NADC30 and the recently reported
NADC30-like strain, CHsx1401, in China (Zhou et al.,
2015), they shared 96.7–99.2% and 95.8–99.2% AA
similar-ity respectively. The AA sequences of the 66 isolates were
analysed in comparison with representative strains (Fig 5),
revealing that AA substitutions were mainly located in the
putative signal peptide and extravirion regions. The regions
from AAs 40–57, 67–90, 107–120 and 138–160 were
rela-tively conserved (Zhou et al., 2009a).

The primary neutralizing epitope (PNE) at AA37–44 of

GP5 and epitope V27LVN are important in inducing
immune responsiveness (Ostrowski et al., 2002).
Com-pared with the VR2332 strain, all of the isolates had a L39I
mutation, and in subgenotype IV, group II, strain
GDSZF1-5 had an S37C mutation. Among the 66 isolates,
only GDZQF3-5 of subgenotype IV group II had an L28P
mutation.

Ansari et al. (2006) found four potential N-glycosylation
sites, N30, N34, N44 and N51, in the extravirion sequence
of GP5. These residues are important for viral infection,
antigen characteristics and susceptibility of the virus to a
neutralizing antibody in vitro. Among the four potential
N-glycosylation sites, the N34 site is prone to mutation,
whereas N44 and N51 are highly conserved in the American
and European type strains (Ansari et al., 2006).
Interest-ingly, all of the isolates had a conserved N30 glycosylation
site. In subgenotype IV group I, only GDSZF1-1, -3 and -4
possessed an N34 glycosylation site. In subgenotype IV
group III, eight isolates had an N34 glycosylation site. All
of the isolates in subgenotype IV group II also had an N34
glycosylation site. N44 and N51 were conserved in all
iso-lates, which is consistent with the report from Ansari et al.
(2006).

Discussion

Porcine reproductive and respiratory syndrome remains a
globally important disease of swine and leads to substantial
economic losses in the pig industry (Lunney et al., 2010;

Shi et al., 2010). In 2006, there was an HP-PRRS outbreak
in south China, caused by an HP-PRRSV strain
character-ized by a 30 AA deletion in the Nsp2 coding region (Li
et al., 2007). In subsequent years, this HP-PRRSV became
the dominant strain in the field, despite low pathogenic
strains also coexisting on pig farms, and type 1 PRRSV
strains also emerging in China (Zhou et al., 2009a). In
recent years, a new mutated strain, NADC30-like, was
reported, indicating further extensive mutations in PRRSV
in China (Zhou et al., 2015). To date, many farmers have
used cell-attenuated modified live vaccine to prevent the
disease, which may increase the immune selective pressure
in pigs and significantly accelerate the variation in PRRSV.

Boars are an important part of the pig production chain
and consequently play a vital role in the spread of PRRSV
(Nathues et al., 2014, 2016); nevertheless, there have been
no reports of PRRSV in boars until now. To fully
under-stand the molecular epidemiology and antibody levels of
PRRSV in boar in South China, we collected 367 field
sam-ples from different pig farms in the Guangdong, Fujian,
Zhejiang and Guangxi provinces of South China in 2015,
the majority of which were from Guangdong. The Nsp2
HV region and ORF5 gene were amplified from the 66
PRRSV-positive samples and sequenced. The rates of
sam-ple positivity differed among regions. The overall positive
rate was 17.25%, whereas the positive rate for Guangdong
was 21.57%; however, samples from Fujian, Zhejiang and
Guangxi were negative, indicating severe PRRS infections
in boars in the Guangdong province of South China.

Fur-thermore, the rates of sample positivity also varied among
the regions within Guangdong, with 13.51%, 7.55%,
Table 3. Information of detected samples in South China

Area City Farm Number %Antigen (n/N)

Pearl River Delta GDGZ F1 34 41.18 (14/34)

GDGZ F2 31 64.52 (20/31)

GDGZ F3 9 0.00 (0/9)

GDHZ F1 9 0.00 (0/9)

GDHZ F2 12 0.00 (0/12)

GDHZ F3 9 0.00 (0/9)

GDJM F1 9 22.22 (2/9)

GDJM F2 3 0.00 (0/3)

GDJM F3 6 33.33 (2/6)

GDZQ F1 1 0.00 (0/1)

GDZQ F2 1 0.00 (0/1)

GDZQ F3 17 29.41 (5/17)

GDZS F1 20 0.00 (0/20)

GDSZ F1 30 23.33 (7/30)

East Guangdong GDMZ F1 3 0.00 (0/3)

GDMZ F2 6 0.00 (0/6)

GDMZ F3 11 27.27 (3/11)

GDHY F1 6 0.00 (0/6)

GDHY F2 11 18.18 (2/11)

West Guangdong GDYJ F1 8 0.00 (0/8)

GDYJ F2 15 0.00 (0/15)

GDMM F1 3 0.00 (0/3)

GDMM F2 3 0.00 (0/3)

GDYF F1 14 0.00 (0/14)

GDZJ F1 10 40.00 (4/10)

North Guangdong GDSG F1 3 0.00 (0/3)

GDQY F1 22 31.82 (7/22)

Fujian FJNP F1 12 0.00 (0/12)

FJFZ F1 10 0.00 (0/10)

Zhejiang ZJHZ F1 13 0.00 (0/13)

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Fig. 3. Phylogenetic tree of PRRSV field isolates based on the translated
amino acid sequence of the Nsp2 HV region The different subgroups
are marked and the PRRSV isolates are labelled with black solid circles.

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26.18% and 28% in east Guangdong, west Guangdong, the
Pearl River Delta and north Guangdong respectively. These
results indicated that the Pearl River Delta and north
Guangdong had higher rates of PRRSV infection. The

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Guangdong. The Pearl River Delta (58%) and north
Guangdong (85.71%) had high LP-PRRSV infection rates.
The overall positive rate for HP-PRRSV and LP-PRRSV
were 10.46% and 11.11% in Guangdong, and 8.72% and
9.26% in South China respectively. Overall, these results
indicate a high infection rate of PRRSV in boars from
South China. The Nsp2 gene naturally contains deletions in
the HV region, resulting in extensive polymorphisms and is
often used for phylogenetic construction and molecular
epidemiology research (Han et al., 2006). Among the 66
Nsp2 sequences that we amplified, four samples
(GDQYF1-3, -4, -5 and -7) had no AA deletions, while all of the others
had varying deleted residues. GDQYF1-3 and -4 belonged
to subgenotype II group I, and shared a great similarity to
the recently reported PRRSV strains, GM2 and QYYZ.

GM2 is a recombinant between the QYYZ field strain and
the MLV RespPRRS/Repro vaccine strain (Lu et al., 2012).
GDQYF1-5 and -7 were highly similar to the LP-PRRSV
strain, SHB, which is endemic in Guangdong. These results
indicate that PRRSV has undergone extensive evolution,
and the fact that different strains exist on the same boar
farm has the potential to lead to recombination and a
resul-tant threat to pig production. Although Zhou et al.
(2009b) reported that the 30-AA deletion in Nsp2 was not
associated with the virulence of the emerging HP-PRRSV,
it has been used as an epidemiological marker,
characteris-tic of the dominant PRRSV strains in China since 2006.
Among the other 62 isolates, 30 had 30 AAs deleted (AAs
482 and 533–561) and belonged to subgenotype III. In
recent years, atypical PRRSV strains have also been
reported (Li et al., 2010; Du et al., 2012), with novel AA
deletions and insertions in Nsp2 (Zhou et al., 2014;
Wang et al., 2015). The two samples GDSZF1-1 and
GDSZF1-3 had a 19-AA deletion of AA533–552 and
shared homology with LP-PRRSV, HB-1-sh-2002 and
HB-1-3.9, while belonging to the HP-PRRSV
representa-tive cluster in subgenotype III. These results further
sup-port the conclusion that the 30-AA deletion in the
Nsp2-coding region is no longer a definitive molecular marker
for Chinese HP-PRRSV (Zhou et al., 2014). Thirty
sam-ples had a 9-AA deletion (AA481–489) and belonged to
the LP-PRRSV representative strain cluster in
subgeno-type II, which was unlike any of the reference strains.
Surprisingly, we found that GDSZF1-2, -4 and -7 had an
8-AA deletion (AA478–485), while on the same boar

farm, isolates GDSZF1-5 and -6 had an obvious 30-AA
deletion. These results suggest that more than one strain
exist on the same boar farm and further imply that
PRRSV has undergone a sizable mutation. Li et al. (2011)
found that the PRRSV strains in Hubei province of
Cen-tral China and Guizhou province of South China
con-tained a discontinuous 59-AA deletion; however, we did
not identify this phenomenon.

The GP5 is one of the most variable structural proteins
of PRRSV and has often been used as a target for analySing
genetic mutations in the virus (Murtaugh et al., 1995; Cha
et al., 2004). Our results demonstrate that all 66 positive
samples belong to the North American genotype and they
could be further divided into two subgenotypes by AA
sequence alignment of GP5. The GDQYF1-5 and -6
belonged to subgenotype III and were highly similar to the
LP-PRRSV-representative strains, SHB and HB-1-3.9.
Other isolates belonged to subgenotype IV and shared high
similarity with the HP-PRRSV cell-attenuated live vaccine
strains, GD-100, JXA1-P80 and TP-90, indicating that
iso-lates in subgenotype IV are probably related to the
exten-sive use of the MLV. We also found that some positive
samples belonged to different subgenotypes when the
phy-logentic trees were constructed based on nsp2 and GP5
respectively. The GDQYF1-3 and -4 had a high similarity
with GD-100, GDQYF1-7 had close homology with
TP-P90, which were also with the HP-PRRSV cell-attenuated
live vaccine strains. These results imply that frequent
recombination between different PRRSV strains existed

and the massive use of vaccines may contribute to this
phe-nomenon.

The PNE of GP5 and epitope V27LVN play important
roles in neutralizing activity and immune responsiveness
(Li et al., 2009). Compared with the VR2332 strain, all
the isolates in this study had an L39I mutation, and in
subgenotype IV group II, GDSZF1-5 had an S37C
sub-stitution. Our initial results indicated that GDSZF1-5
was a HP-PRRSV; therefore, these results indicate that
the AA substitution in the PNE may be a major
contrib-utor to PRRSV escape of immune defences and the virus
epidemic on the boar farm in Shenzhen, Guangdong.
Among all samples, only GDZQF3-5 of subgenotype IV
group II had an L28P mutation. Due to the vital role of
the PNE epitope and V27LVN (Ostrowski et al., 2002),
these critical AA substitutions could contribute to the
failure of immune protection and escape of the virus
from neutralization, leading to inefficacy of
cell-attenu-ated live vaccine strains, such as the CH-1R and the
JXA1-P80 vaccine strains (derived from LP-PRRSV strain
CH-1a and HP-PRRSV strain JXA1). Four potential
N-glycosylation sites, N30, N34, N44 and N51, in the
extravirion of GP5 are related to viral infection
parame-ters, including antigen characteristics and the
susceptibil-ity of the virus to a neutralizing antibody in vitro

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GDSZF1-1, -3 and -4 possessed an N34 glycosylation
site, while it was present in eight isolates of subgenotype
IV group III. N44 and N51 were also conserved in all

isolates. These results demonstrate that the four potential
N-glycosylation sites are reduced in the samples in our
study, which is consistent with the results of Xie et al.,
(2013), who has investigated the epidemiology of PRRSV
in South China, but is contrary to the findings of Li
et al. (2011). A previous study confirmed that abolition
of the N-glycosylation sites in GP5 could increase the
ability of the mutant virus to resist neutralization
(Bar-foed et al., 2004); therefore, the observed decrease in the
number of potential N-glycosylation sites may explain
the continuous mutation of PRRSV and the failure of
vaccine protection.

In this study, we performed the first comprehensive
investigation of the epidemiology of boars infected with
PRRSV in South China by molecular sequence alignment
and phylogenetic analysis. Our results demonstrate the
existence of multiple different strains on the same farm and
extensive genetic mutation of PRRSV in boars. These
results will be useful in understanding the epidemiology of
PRRS on boar farms and in developing a series of measures
to control this disease.

Acknowledgements

This work was supported by the National Key Technologies
R&D Program (2015BAD12B02-5) and Guangzhou City
Project (201508020062).

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