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Veterinary Microbiology 158 (2012) 291–299

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Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic

New genomic characteristics of highly pathogenic porcine reproductive and respiratory syndrome viruses do not lead to significant changes in pathogenicity Xiuling Yu a,1, Nanhua Chen a,b,1, Lilin Wang a, Jiajun Wu a, Zhi Zhou a, Jianqiang Ni a, Xiangdong Li b, Xinyan Zhai a, Jishu Shi b, Kegong Tian a,* a b

Veterinary Diagnostic Lab, China Animal Disease Control Center, No. 2 Yuanmingyuan West Rd., Haidian District, Beijing 100193, PR China Department of Anatomy and Physiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506, USA

A R T I C L E I N F O

A B S T R A C T

Article history: Received 27 December 2011 Received in revised form 16 February 2012 Accepted 23 February 2012

Highly pathogenic porcine reproductive and respiratory syndrome (HP-PRRS) initially emerged in China and currently prevails in other Asian countries as well, resulting in immense economic losses. HP-PRRS virus (HP-PRRSV) has undergone rapid evolution since its first recognition in 2006. To analyze the genomic and pathogenic characteristics of 2010 HP-PRRSV, we tested 919 clinical samples collected from China, Laos and Vietnam, sequenced 29 complete genomes of HP-PRRSV isolates, and determined the pathogenicity of seven HP-PRRS viruses isolated from 2006 to 2010. HP-PRRSV was detected from 45.2% (415/919) samples, while only 0.1% (1/919) was classical PRRSV, indicating that HP-PRRSV isolates with a unique discontinuous deletion of 30 amino acids (aa) in non-structural protein 2 (Nsp2) are still the predominant viruses. 2010 HP-PRRSV together with 2009 HPPRRSV isolates form a new evolutionary branch based on phylogenetic analyses. The numbers of potential N-glycosylation sites are variable in major glycoprotein GP5 but are conserved in minor glycoproteins GP2, GP3 and GP4. Pathogenicity studies showed that HP-PRRS viruses isolated from 2006 to 2010 maintain similar level of high pathogenicity, which caused high fever (>41 8C for at least four days), 100% morbidity, and 40–100% mortality in 4–10 weeks old pigs. Real time monitoring information from this study could help to understand the genetic and pathogenic evolution of HP-PRRSV and assist in the control of HP-PRRS in Asia. ß 2012 Published by Elsevier B.V.

Keywords: HP-PRRSV Evolution Genome Pathogenicity Epidemiology

1. Introduction Porcine high fever disease (PHFD), characterized by high fever (40–42 8C), abnormally high morbidity (50– 100%) and mortality (20–100%) in all ages of pigs, first emerged in Southern China in 2006 and currently is almost ubiquitous throughout China (Li et al., 2007; Tian et al.,

* Corresponding author at: Veterinary Diagnostic Lab, China Animal Disease Control Center, No. 2 Yuanmingyuan West Rd., Haidian District, Beijing 100193, PR China. Tel.: +86 10 62891257; fax: +86 10 62893507. E-mail address: [email protected] (K. Tian). 1 These authors contributed equally to this work. 0378-1135/$ – see front matter ß 2012 Published by Elsevier B.V. doi:10.1016/j.vetmic.2012.02.036

2007; Tong et al., 2007). According to statistical data from the China Animal Disease Control Center (CADC), PHFD epidemics affected more than 2,000,000 pigs and resulted in the death of at least 400,000 pigs in 2006 (Tian et al., 2007). The recurrence of PHFD affected more than 140,000 pigs and caused the death of about 40,000 pigs in 2007 (Chen et al., 2009). In 2008, only sporadic outbreaks were reported, which affected 7648 pigs with 2908 fatal cases. However, PHFD reemerged in 2009 with over 1,000,000 cases of infection (Zhou et al., 2011). The latest PHFD epidemic in 2010 affected 17,259 pigs and killed 5593 pigs. This new infectious disease has resulted in immense economic losses for the Chinese swine industry, and the

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pronounced decline of swine production caused by PHFD is closely correlated with the dramatically rising pork price in China in the last five years. More importantly, PHFD epidemics has subsequently erupted in Southeast Asia and resulted in destructive impacts on the local pig husbandries since 2007 (An et al., 2011; Barrette et al., 2009; Dietze et al., 2011; Feng et al., 2008; Metwally et al., 2010; Normile, 2007; Roberts et al., 2009). Although PHFD is a significant problem for swine industries in Asia at the present time, it may become a huge threat to swine production all over the world in the near future. In 2007, highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV), which is a novel mutant of PRRSV containing a unique discontinuous deletion of 30 amino acids (aa) in non-structural protein 2 (Nsp2), was identified to be the etiological agent of PHFD (Tian et al., 2007). PRRSV, an enveloped, single-stranded positive-sense RNA virus with an approximately 15 kb genome possessing a 50 -capped structure, a 30 -polyadenylated tail and ten open reading frames (ORFs) flanked by 50 and 30 untranslated regions (UTR), has been clustered in the family Arteriviridae within the order Nidovirales (Cavanagh, 1997; Firth et al., 2011; Johnson et al., 2011; Snijder and Meulenberg, 1998). PRRSV was initially recognized in North America in 1987, in Western Europe in 1990, and then subsequently spread to Asia (Albina, 1997; Keffaber, 1989; Wensvoort et al., 1991). Although PRRSV isolates identified around the world cause similar disease symptoms and share the same virion morphology, they are antigenically, genetically, and pathologically very heterogenic (Murtaugh et al., 1995). Thus far, two genotypes of PRRSV have been defined: the European strain (EU genotype, type I) and the North American strain (NA genotype, type II), with Lelystad virus (LV) and ATCC VR-2332 strain serving as prototypic viruses (Meng et al., 1995; Nelsen et al., 1999), respectively. Type II PRRSV was first recognized in mainland China at the end of 1995 in Beijing (Guo et al., 1996). Since then it has become widespread and predominant, although type I PRRSV has also been isolated in China in recent years (Chen et al., 2011; Li et al., 2010a). HP-PRRSV is a new variant of type II PRRSV and probably originated from CH-1a, which is the first PRRSV isolated in China (An et al., 2010). HP-PRRSV is a rapidly evolving virus and two of its most variable genes, Nsp2 and ORF5, are usually chosen for evolution and divergence analyses (Chen et al., 2011; Normile, 2007; Zhou et al., 2009a, 2011). More than 100 complete genomes and over 1,000 partial sequences of HPPRRSV have been published in the last five years. Some novel mutants were identified and isolates with different virulence were also reported (Li et al., 2010b; Tian et al., 2007; Zhou et al., 2011; Zhu et al., 2011). Whether the genetic and pathogenic evolutions of HP-PRRSV are completely erratic or relatively regular and constant has not been previously reported. The widespread epidemic, persistent recurrences, and significant losses due to this intractable disease call for real time monitoring the disease and the evolutionary trend of HP-PRRSV. In this study, we report the up-to-date epidemiology of HP-PRRS, and analyze the genetic and pathogenic evolutions of HPPRRSV isolated in China, Laos and Vietnam.

2. Materials and methods 2.1. Epidemiological investigation During January to December in 2010, sera and tissue samples from swine farms in mainland China were collected by the China Animal Disease Control Center (CADC). In addition, clinical samples from one farm in Vietnam and eight farms and one slaughter house in Laos were also submitted to CADC for differential diagnosis. A conventional RT-PCR assay for both genotypes of PRRSV was used to test a total of 919 samples from 919 pigs (one sample was collected from each pig) and a duplex realtime RT-PCR assay was applied to all PRRSV positive samples for rapid differential detection (Chen et al., 2009). Samples with co-infection of other swine viruses such as classical swine fever virus, porcine circovirus type 2, porcine parvovirus and pseudorabies virus were excluded from this study for further analysis. 2.2. Genomic sequencing analyses of HP-PRRSV We selected 29 isolates according to their geographic distribution as representative viruses for complete genomic sequencing, including 21 from China, seven from Laos and one from Vietnam. Specifically, these clinical samples were collected from Beijing, Hebei, Shandong, Jilin, Jiangxi, Fujian and Guangxi provinces in China, the capital Vientiane of Laos and the Quang Nam province in Vietnam. RNA was extracted from the clinical samples with RNeasy Mini Kit (Qiagen, Germany) according to manufacturer’s instructions. 18 pairs of primers amplifying 18 overlapped fragments of type II PRRSV were used for full-length sequence determination (Han et al., 2009). The RT-PCR systems and amplification conditions have been previously described (Zhou et al., 2011). The amplicons were purified with an E.Z.N.A. Gel Extraction Kit (OMEGA, Norcross, GA, USA) and cloned into pGEM-T Easy vector (Promega, Madison, WI, USA). The recombinant clones were sequenced by an ABI PRISM 3730 sequencer (Applied Biosystems, Foster, CA, USA) using specific primers for the T7 and SP6 promoters in both directions. Each fragment was independently sequenced at least three times. 2.3. Sequence alignments and phylogenetic analyses There were 34 complete genomes determined during 2006 and 2010 by our laboratory and 11 representative full-length sequences obtained from GenBank that were utilized in phylogenetic analyses (Table 1). Three phylogenetic trees were constructed based on complete genomes, Nsp2 genes or ORF5 genes of the 45 PRRSV isolates. Multiplex sequence alignments were generated by CLUSTAL X (version 1.83) (Jeanmougin et al., 1998). Phylogenetic analyses were conducted using the MEGA software (version 4.0) with the neighbor-joining method, the maximum composite likelihood model and the bootstrap confidence value of 1000 replicates (Tamura et al., 2007). Other detailed parameters used for constructing the phylogenetic trees in this study were the same as in our previous report (Chen et al., 2011).

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Table 1 PRRSV isolates used in this study.* No.

Designed name

Year isolated

Countries of origin

Accession no.

No.

Designed name

Year isolated

Countries of origin

Accession no.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

VR-2332 BJ-4 CH-1a HB-1 HB-2 NB/04 GD3 JXA1 HuN4 07BJ Shaanxi-2 07QN 08HuN YN9 SX2009 09HUB1 10BJ-1 10BJ-2 10BJ-3 10BJ-4 10BJ-5 10FUJ-1 10FUJ-2

1992 1996 1996 2002 2002 2004 2005 2006 2006 2007 2007 2007 2008 2008 2009 2009 2010 2010 2010 2010 2010 2010 2010

USA China China China China China China China China China China Vietnam China China China China China China China China China China China

PRU87392 AF331831 AY032626 AY150312 AY262352 FJ536165 GU269541 EF112445 EF635006 FJ393459 HQ401282 FJ394029 GU169411 GU232738 FJ895329 JF268682 JQ663541 JQ663543 JQ663542 JQ663544 JQ663545 JQ663546 JQ663547

24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45

10FUJ-3 10FUJ-4 10FUJ-5 10GX-1 10GX-2 10GX-3 10GX-4 10GX-5 10HEB-1 10HEB-2 10HEB-3 10JL 10JX 10SD LW1-13 LW2-6 LW3-7 LW5-1 LW6-6 LW7-1 LW8-1 10QN

2010 2010 2010 2010 2010 2010 2010 2010 2010 2010 2010 2010 2010 2010 2010 2010 2010 2010 2010 2010 2010 2010

China China China China China China China China China China China China China China Laos Laos Laos Laos Laos Laos Laos Vietnam

JQ663548 JQ663549 JQ663550 JQ663558 JQ663559 JQ663560 JQ663561 JQ663562 JQ663551 JQ663552 JQ663553 JQ663554 JQ663540 JQ663555 JQ663557 JQ663563 JQ663564 JQ663565 JQ663566 JQ663567 JQ663568 JQ663556

* The genomes determined by our lab are bold.

2.4. Pathogenicity evaluations Animal infection experiments were performed using 60 PRRSV-free piglets from four to ten weeks old in three studies during 2008, 2009 and 2010. These pigs are also free of other swine pathogens such as classical swine fever virus, swine influenza virus, porcine circovirus type 2, porcine parvovirus, pseudorabies virus and porcine torque teno virus. In each experiment, 20 pigs of the same age were randomly divided into four groups (five in each group) and three groups were challenged with corresponding viruses (JXA1, HEBTJ, and 08CQ; or JXA1, 09SC, and 09JL1; or JXA1, 10BJ-5, and LW1-13), while one group was mock infected with Dulbecco’s Modified Eagle Medium (DMEM) as the negative control. The groups challenged with JXA1 were used as the positive control. The virus load of intramuscular injection was 3.0 mL of 104.5 TCID50/mL virus culture. Pigs were maintained separately for three weeks and monitored daily for rectal temperature and clinical signs. Surviving pigs were euthanized at 21 days post challenge (dpc). Animal experiments in this study were approved by the CADC ethics committee (Permit numbers CADC-AEC-2008021, CADC-AEC-2009007 and CADC-AEC-2010008). 3. Results 3.1. 2010 HP-PRRS epidemics are prevalent in China and Southeast Asia Among the 919 swine clinical samples collected by the China Animal Disease Control Center (CADC) from swine farms and slaughter houses in China, Vietnam and Laos in 2010, 415 (45.2%) of these samples were diagnosed as HPPRRSV positive, only one (0.1%) of them was classical type II PRRSV positive while none was type I PRRSV positive

through differential detection. Based on epidemiological analyses of our data collected constantly from 2006 to 2010, HP-PRRSV existed in all provinces of mainland China and became the predominant isolates. What is more, the identification of HP-PRRSV positive clinical samples collected from Laos and Vietnam in 2010 demonstrated the emergence and reemergence of HP-PRRS in these countries, respectively. Fig. 1 was constructed with our research and previous reports (An et al., 2011; Barrette et al., 2009; Dietze et al., 2011; Feng et al., 2008; Metwally et al., 2010; Normile, 2007; Roberts et al., 2009; Tian et al., 2007) to present the emergence and spreading tendency of HP-PRRSV epidemics. HP-PRRS epidemics initially emerged in China in 2006, and were subsequently confirmed in Southeast Asian countries including Vietnam (2007), Philippines (2007), Thailand (2009), Cambodia (2010) and Laos (2010). In addition, HP-PRRS-like diseases were also reported in neighboring countries such as Bhutan, Burma, Malaysia, Singapore, South Korea and Russia (An et al., 2011; Dietze et al., 2011). Currently, HPPRRS has become a significant threat for the Asian swine industry and continues to spread worldwide. 3.2. 2010 HP-PRRSV isolates share high genetic identity and retain unique genetic marker All the 29 latest HP-PRRSV showed high similarity with HP-PRRSV reference strain JXA1. The 21 Chinese isolates shared 97.9% to 98.5% nucleotide identity with JXA1 while the identities were 97.6% to 100% among themselves. The identities of Laotian isolates and JXA1 were between 98.0% and 98.1% while they shared 99.8% to 99.9% identity themselves. Vietnam isolate 10QN had 99.2% identity with JXA1 and 98.6% identity with the 2007 Vietnam isolate 07QN (Feng et al., 2008). In addition, all the 2010 HPPRRSV isolates from China, Laos and Vietnam still had the

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Fig. 1. HP-PRRS epidemics are prevalent in Asia between 2006 and 2010. In this geographical and temporal map, HP-PRRSV positive countries are marked with different colors based on the year in which the first HP-PRRSV was identified in that country (2006 in red, 2007 in pink, 2009 in blue, 2010 in light blue). Countries with HP-PRRS-like epidemics are shown in gray. This map is modified from an Asia map (http://www.mwcsk12.org/elementary/continents.htm). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

HP-PRRSV genetic marker, a unique 30 amino acid discontinuous deletion at position 481 and 533 to 561. No new deletions or insertions were identified (data not shown). 3.3. 2010 HP-PRRS viruses are similar to 2009 isolates but are different from 2006 to 2008 isolates To study the genetic evolutionary trend of 2010 HPPRRSV isolates, we constructed phylogenetic trees based on 29 isolates of 2010 HP-PRRSV, 15 representative PRRS viruses isolated in China and Vietnam from 1996 to 2009, and the type II reference strain VR-2332. Consistent with previous reports (An et al., 2010; Li et al., 2011, 2010a; Zhou et al., 2011), Chinese type II PRRSV can be divided into four groups: VR-2332-like isolates, CH-1a-like classical isolates, HB-1-like intermediate isolates and JXA1-like highly pathogenic isolates. All the 2010 HP-PRRSV isolates were clustered in the farthest evolutionary branch, had the highest similarity with 2009 HP-PRRSV isolates, but were

significantly different from 2006 to 2008 HP-PRRSV isolates (except 10QN which was in the branch of 2006– 2008 HP-PRRSV isolates in the phylogenetic tree based on complete genome) (Fig. 2A–C). Therefore, the latest HPPRRSV in China, Laos and Vietnam were the products of continuing evolution of HP-PRRSV and the formation of a new evolutionary subgroup after 2009 by the isolates that were genetically different from 2006 to 2008 HP-PRRSV isolates. 3.4. Potential N-glycosylation sites are variable in GP5 but are conserved in GP2, GP3 and GP4 To gain further insight into the genetic evolution of the latest HP-PRRSV in the environment of widespread HPPRRSV infection and vaccination, we analyzed the changes of potential N-glycosylation sites (sequence NXS/T, where X is any amino acid except Pro and Asp) in both the major envelope glycoprotein GP5 and minor glycoproteins GP2, GP3 and GP4. As shown in Fig. 3, the numbers of potential

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Fig. 2. Phylogenetic analyses show that HP-PRRSV isolates after 2009 formed a new subgroup that differed from 2006 to 2008 HP-PRRSV isolates. Three phylogenetic trees were constructed based on complete genomes (A), Nsp2 genes (B), and ORF5 genes (C) of 45 PRRSV isolates. VR-2332-like isolates are labeled in red, CH-1a-like isolates in purple, HB-1-like isolates in light blue, JXA1-like isolates in green and the new SX2009-like isolates in the furthest branch are marked in blue. Each isolate is indicated by the year of isolation and the designated name (e.g. 06 JXA1 means that JXA1 was isolated in 2006). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

N-glycosylation sites in GP5 of Chinese PRRSV varied from three to five. Most of the HP-PRRSV and intermediate PRRSV gained a new N-glycan moiety at position 35 (except 10FUJ-3, 10GX-4 and 10GX-5 for HP-PRRSV and HB-1 for intermediate PRRSV) when compared with VR2332 and classical Chinese isolates. More interestingly, most of the 2009 and 2010 HP-PRRSV isolates lost the Nglycosylation site at position 34 (except 10JL) when compared with 2006 to 2008 HP-PRRSV isolates. The other three N-glycosylation sites N30, N44 and N51 were highly conserved in all PRRSV except the N51S mutation in the 2007 isolates Shaanxi-2 and the N30D mutation in the 2010 isolates 10FUJ-1. In addition, the L39I mutation within the major neutralizing epitopes (from aa 37 to 44) presented in all HP-PRRSV isolates. In the decoy epitopes (between aa 27 and 31), the A29V mutation was found in 2006 to 2008 HP-PRRSV isolates and most of the classical and intermediate Chinese PRRSV isolates, but was mutated from V29 back to A29 as seen in VR-2332 for 2009 and 2010 HPPRRSV isolates. In contrast to the variability of N-glycan moieties in GP5, potential N-glycosylation sites in minor glycoproteins were relatively conserved in PRRSV. GP2 had only one

potential N-glycosylation site at position 181, which was highly conserved in all PRRSV isolates. The number of glycan moieties in GP3 was between six and seven; all HPPRRSV isolates and majority of Chinese classical and intermediate PRRSV isolates (except BJ-4 and GD3) had one more N-glycosylation site at position 27 when compared to VR-2332. GP4 of all PRRSV isolates had the same four Nglycosylation sites although JXA1, 10SD and 10QN had the mutation T132S within the potential glycosylation site. 3.5. Predominant HP-PRRSV isolates maintained high pathogenicity over the past five years To determine the pathogenic changes of HP-PRRS viruses isolated from 2006 to 2010, seven temporally and geographically representative HP-PRRSV isolates, including JXA1, HEBTJ, 08CQ, 09SC, 09JL1, 10BJ-5 and LW1-13, were utilized in three animal infection studies. Three animal experiments were conducted in three different years: 2008, 2009, and 2010. To ensure a meaningful comparison among the isolates from 2008 to 2010, we selected JXA1, a HP-PRRS virus isolated in China Jiangxi Province in 2006, as a positive control for HP-PRRS

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Fig. 3. Potential N-glycosylation sites are variable in GP5 of HP-PRRS viruses isolated in China, Laos and Vietnam from 2006 to 2010. Sequences from representative type II strains, such as VR-2332, BJ-4, CH-1a, HB-1, HB-2, NB/04 and GD3, are also used for comparison. The dashed box indicates the decoy epitope domain. The solid box indicates the primary neutralizing epitope domain. The potential N-glycosylation sites are highlighted in gray. Most of the 2009 and 2010 HP-PRRSV isolates lost the N-glycosylation site at position 34 (except 10JL) when compared with 2006–2008 HP-PRRSV isolates. The novel mutations resulting in loss of N-glycan moiety are asterisked.

virus in each of the experiment. As shown in Table 2, compared to healthy pigs in negative control groups, all pigs infected with different HP-PRRSV isolates showed high fever (41 8C) at two to six days post challenge

(dpc) that persisted for at least four days thereafter. In addition, obvious clinical symptoms including skin cyanopathy, lethargy, anorexia, ataxia and dyspnea were observed in all challenged groups. Although 100% of the

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297

Table 2 Pathogenicity comparison of seven HP-PRRS viruses isolated from 2006 to 2010.a Studies (Year)

Isolates

Years

Locations

Passagesb

Virus titers (TCID50/mL)

High fever (41 8C)

Morbidityc (%)

1st (2008)

JXA1 HEBTJ 08CQ

2006 2007 2008

Jiangxi, China Tianjin, China Chongqing, China

5th 7th 8th

104.5 104.5 104.5

7 7 7

Yes Yes Yes

100 100 100

80 40 40

2nd (2009)

JXA1 09SC 09JL1

2006 2009 2009

Jiangxi, China Sichuan, China Jilin, China

5th 5th 5th

104.5 104.5 104.5

4 4 4

Yes Yes Yes

100 100 100

100 100 100

3rd (2010)

JXA1 10BJ-5 LW1-13

2006 2010 2010

Jiangxi, China Beijing, China Vientiane, Laos

5th 5th 5th

104.5 104.5 104.5

10 10 10

Yes Yes Yes

100 100 100

40 40 60

Ages of pigs (weeks)

Mortality (%)

a Each group (5 piglets) was challenge with one virus. Clinical monitoring was conducted daily for 21 days and then pigs were euthanized for pathological and histopathological analysis. b Different viral passages were used so that each virus preparation reached the same virus titer (104.5 TCID50/mL). c Signs of morbidity: (1) high fever for at least 3 days; (2) respiratory symptoms; (3) lung consolidation.

HP-PRRSV-infected pigs developed clinical symptoms, mortality rates ranged from 40% to 100%. This could be correlated with differences in the age of pigs (from 4 to 10 weeks old) and/or the passage number of viruses (from 5th to 8th) used in these experiments. Pathological changes such as lung edema, hemorrhagic spots in lung and kidney, and hemorrhages and necrosis in lymph nodes were observed in challenged pigs upon necropsy at 21 dpc. Visible interstitial pneumonia characterized by atrophy of alveoli and proliferation and infiltration of lymphocytes could be observed in infected pigs histopathologically (data not shown). Overall, 2007–2010 HP-PRRSV isolates have similar pathogenicity as the 2006 isolate JXA1. All HP-PRRSVinfected pigs exhibited obvious clinical signs, persistent high fever, 100% morbidity and 40–100% mortality. The results indicated that the predominant HP-PRRSV isolates still maintained high pathogenicity over the five-year time span and new genetic characteristics obtained by 2009 and 2010 isolates did not lead to significant changes in pathogenicity compared with HP-PRRSV isolated from 2006 to 2008. 4. Discussion We have investigated the emergence and reemergence, transmission and spreading tendency of HP-PRRSV through epidemiological survey, genomic sequencing, phylogenetic analysis, and pathogenicity evaluation of various HP-PRRS viruses isolated in China, Vietnam and Laos from 2006 to 2010. Our data showed that HP-PRRS viruses with the typical genetic marker of a 30 aa discontinuous deletion in Nsp2 are still the predominant isolates. Most of the HP-PRRS viruses isolated between 2009 and 2010 were genetically different from the viruses isolated from 2006 to 2008 and formed a new evolutionary branch. However, most of the HP-PRRSV isolates maintained the characteristic of high pathogenicity over the past five years even though they gained new genomic characteristics through continuing evolution. PRRSV is one of the most rapidly evolving viruses and its evolutionary rate of 4.7–9.8  102/site/year is the highest among RNA viruses reported so far (Hanada et al., 2005;

Normile, 2007). HP-PRRSV, which has evolved from Chinese type II PRRSV principally through mutations (An et al., 2010), is characterized by the 30 aa discontinuous deletion within Nsp2 (Tian et al., 2007), even though the Nsp2 deletion is likely to be a genetic marker rather than a virulence determinant (Han et al., 2009; Ni et al., 2011; Zhou et al., 2009b). During the past five years, although several HP-PRRSV isolates with novel deletions within Nsp2 were reported (Li et al., 2009; Zhou et al., 2011; Zhu et al., 2011), HP-PRRS viruses with the unique 3a discontinuous deletion in Nsp2 were still the predominant isolates (Li et al., 2011, 2010a; Zhou et al., 2011). All of the 29 viruses isolated in 2010 from China, Laos and Vietnam maintained the unique genetic marker in Nsp2. However, most of them are clustered in the farthest branch from 2006 isolates and show the closest relationship with 2009 isolates according to our phylogenetic analyses. This phenomenon is consistent with our recent report (Zhou et al., 2011) and supports the hypothesis that HP-PRRSV isolates after 2009 obtained new genetic properties and formed a new subgroup during the course of evolution. Chinese swine herds have widespread HP-PRRSV infection and coexistence with classical type II PRRSV and type I PRRSV (Chen et al., 2011; Li et al., 2011). Furthermore, six live attenuated PRRSV vaccines, including Ingelvac PRRS MLV, CH-1R and R98 for classical type II PRRSV, and JXA1-R, HuN4-F112 and TJM-F92 specific for HP-PRRSV, are marketed in China. The pressure produced by the complicated infection and vaccination condition will significantly facilitate the rapid evolutio0 an of PRRSV, which may result in producing new isolates which can escape host immune responses. Glycan shielding is an important mechanism for PRRSV immune evasion (Ansari et al., 2006; Vu et al., 2011). Glycosylation of both the major and minor viral envelope glycoproteins can help PRRSV to escape, block, or minimize the viral neutralizing antibody response (Vu et al., 2011). In this study, we have found that compared to the increased N-glycan moieties in 2006–2008 HP-PRRSV isolates at position 34 of GP5, the majority of tested 2009 and 2010 HP-PRRSV isolates lost this N-glycosylation site (except 10JL). Furthermore, three 2010 HP-PRRSV isolates (10FUJ-3, 10GX-4 and 10GX-5) also lost the N-glycan moiety at position 35 of GP5.

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In addition, within the decoy epitopes of GP5, the V29 in 2006–2008 HP-PRRSV isolates was substituted by A29 in 2009 and 2010 HP-PRRSV isolates. Two other mutations, N30D for 10FUJ-1 and N51S for Shaanxi-2, were observed in two highly conserved N-glycosylation sites. N-glycan moieties play an important role on virus infectivity and ability to induce neutralizing antibodies (Ansari et al., 2006; Vu et al., 2011). Substitutions could change the neutralizing activity of neutralizing antibodies induced by currently available live attenuated vaccines against the 2010 HP-PRRSV isolates helping new isolates to block or minimize the neutralization and increase the viral infectivity, thus enhancing fitness or survival for the new HP-PRRSV isolates. For the minor glycoproteins, all HP-PRRSV isolates and some Chinese classical and intermediate type II PRRSV isolates had one more N-glycan moiety at position 27 of GP3 than did VR-2332, while other glycan moieties were highly conserved in the minor glycoproteins. This change in GP3 could be correlated with the high immune pressure in Chinese swine herds and might help the isolates gain the capability to escape the neutralization antibodies induced by the vaccines derived from VR-2332. The pathogenicity of JXA1 was first reported by us in 2007 (Tian et al., 2007). Although the pigs used in these experiments were different in age, each of our studies used the same age of pigs and we compared the pathogenicity of 2007–2010 isolates with the same positive control 2006 isolate JXA1. As shown in Table 2, there is no significant pathogenicity difference between 2007 and 2010 isolates and JXA1. They all led to 100% morbidity and 40–100% mortality, which is the same as the pathogenicity of 2006– 2007 isolates reported by us and others (Li et al., 2007; Tian et al., 2007; Tong et al., 2007). Thus, it is reasonable to conclude that the pathogenicity of HP-PRRSV do not significantly change from 2006 to 2010. PRRSV is a unique virus which was first circulated among pigs as a nonpathogenic strain and then gradually evolved into a pathogenic one (Normile, 2007). Since PRRSV originally emerged in China in 1995, it has become widespread and the infection rate can reach up to 90% in Chinese swine herds. In contrast with the classical PRRSV isolates with low pathogenicity, only causing diseases in piglets, but rarely killing grown pigs (Chen et al., 2011; Guo et al., 1996), novel HP-PRRSV isolates not only could result in a high mortality in piglets but also caused the death of grown pigs, even multiparous sows (Tian et al., 2007). During the past five years, several low pathogenicity field isolates which maintained the characteristic 30 aa discontinuous deletion have been identified, such as GDQJ and SY0909 (Li et al., 2010b; Wang et al., 2011). However, consistent with other recent reports (Li et al., 2011, 2010a; Zhou et al., 2009a, 2011), our pathogenicity analysis using geographically and temporally different isolates from China and Laos support the hypothesis that most recent field HP-PRRSV isolates are still highly pathogenic. Furthermore, although 2009 and 2010 isolates gained new genetic properties, that change did not result in significant difference in pathogenicity between 2006– 2008 isolates and 2009–2010 isolates. However, in light of the fact that few naturally attenuated viruses were

discovered, super pathogenic isolates may also emerge in the future due to the fast evolution of HP-PRRSV, which call for the real time monitoring of HP-PRRSV evolution.

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