Bài tập tổng hợp - C4

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[r]

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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

1College of Animal Science & National Engineering Center for Swine Breeding Industry, South China Agriculture University, Guangzhou, China 2Xinyang Animal Disease Prevention and Control Engineering Research Center, Xinyang College of Agriculture and Forestry, Xinyang, China 3Testing 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: cxsong2004@163.com; cxsong@scau.edu.cn

and

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

Fax: 02087038106; E-mail: fanfuhao@139.com

*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 PRRSV (Euro-pean genotype) is represented by the prototype strain Lelys-tad virus, while type 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, ll 59 reverse transcription buffer, 2ll deoxynucleoside triphosphate (dNTP) mixture (10 mM), 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 h, then incubated on ice for 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 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 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 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

6 GDGZF1-6 Guangzhou 603 1035 39 GDMZF3-1 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

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

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

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

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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 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 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 0.00 (0/9)

GDHZ F1 0.00 (0/9)

GDHZ F2 12 0.00 (0/12)

GDHZ F3 0.00 (0/9)

GDJM F1 22.22 (2/9)

GDJM F2 0.00 (0/3)

GDJM F3 33.33 (2/6)

GDZQ F1 0.00 (0/1)

GDZQ F2 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 0.00 (0/3)

GDMZ F2 0.00 (0/6)

GDMZ F3 11 27.27 (3/11)

GDHY F1 0.00 (0/6)

GDHY F2 11 18.18 (2/11)

West Guangdong GDYJ F1 0.00 (0/8)

GDYJ F2 15 0.00 (0/15)

GDMM F1 0.00 (0/3)

GDMM F2 0.00 (0/3)

GDYF F1 14 0.00 (0/14)

GDZJ F1 10 40.00 (4/10)

North Guangdong GDSG F1 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 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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