Accepted Manuscript

Title: Genetic and immunobiological diversity of porcinereproductive and respiratory syndrome genotype I strains

Authors: Laila Darwich, Mariona Gimeno, Marina Sibila, IvanDiaz, Eugenia de la Torre, Silvia Dotti, Liudmila Kuzemtseva,Margarita Martin, Joan Pujols, Enric Mateu

PII: S0378-1135(11)00012-5DOI: doi:10.1016/j.vetmic.2011.01.008Reference: VETMIC 5146

To appear in: VETMIC

Received date: 22-6-2010Revised date: 30-12-2010Accepted date: 10-1-2011

Please cite this article as: Darwich, L., Gimeno, M., Sibila, M., Diaz, I., de laTorre, E., Dotti, S., Kuzemtseva, L., Martin, M., Pujols, J., Mateu, E., Genetic andimmunobiological diversity of porcine reproductive and respiratory syndrome genotypeI strains, Veterinary Microbiology (2010), doi:10.1016/j.vetmic.2011.01.008

This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.

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Title: Genetic and immunobiological diversity of porcine reproductive and respiratory 1

syndrome genotype I strains. 2

3

Authors: Laila Darwich1,2 *ŧ

, Mariona Gimeno1,2ŧ

, Marina Sibila2, Ivan Diaz

2, Eugenia 4

de la Torre2, Silvia Dotti

2¶, Liudmila Kuzemtseva

2, Margarita Martin

1,2, Joan Pujols

2,3, 5

Enric Mateu1,2

6

7

Affiliations: 8

1 Departament de Sanitat i Anatomia Animals, Universitat Autònoma de Barcelona, 9

08193 Bellaterra, Spain. 10

2 Centre de Recerca en Sanitat Animal (CReSA), UAB-IRTA, campus UAB, 08193 11

Bellaterra, Spain. 12

3 Institut de Recerca i Tecnologia Agroalimentària (IRTA), Barcelona, Spain. 13

¶ Current address: Instituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia 14

Romagna "Bruno Ubertini"; Via Bianchi, 9; 25124 Brescia (Italy). 15

ŧ LD and MG have contributed equally to this study. 16

*Corresponding author: Laila Darwich 17

e-mail: laila.darwich@uab.cat 18

Tel #: +34935811046 19

Fax #: +34935813297 20

21

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Summary 22

Genetic diversity of porcine reproductive and respiratory syndrome virus (PRRSV) has 23

been based on ORF5/GP5 and ORF7/N protein variations. Complete viral genome 24

studies are limited and focused on a single or a few set of strains. Moreover, there is a 25

general tendency to extrapolate results obtained from a single isolate to the overall 26

PRRSV population. In the present study, six genotype-I isolates of PRRSV were 27

sequenced from ORF1a to ORF7. Phylogenetic comparisons and the variability degree 28

of known linear B-epitopes were done considering other available full-length genotype-I 29

sequences. Cytokine induction of all strains was also evaluated in different cellular 30

systems. Non structural protein 2 (nsp2) was the most variable part of the virus with 2 31

out of 6 strains harboring a 74 aa deletion. Deletions were also found in ORF3 and 32

ORF4. Phylogenetic analyses showed that isolates could be grouped differently 33

depending on the ORF examined and the highest similarity with the full genome cluster 34

was found for the nsp9. Interestingly, most of predicted linear B-epitopes in the 35

literature, particularly in nsp2 and GP4 regions, were found deleted or varied in some of 36

our isolates. Moreover, 4 strains, those with deletions in nsp2, induced TNF- and 3 37

induced IL-10. These results underline the high genetic diversity of PRRSV mainly in 38

nsp1, nsp2 and ORFs 3 and 4. This variability also affects most of the known linear B-39

epitopes of the virus. Accordingly, different PRRSV strains might have substantially 40

different immunobiological properties. These data can contribute to the understanding 41

of PRRSV complexity. 42

43

Keywords: Porcine reproductive and respiratory syndrome virus; complete ORFs 44

sequences; genotype-I; phylogenetic analyses; immunobiological properties. 45

46

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Introduction 47

Porcine reproductive and respiratory syndrome virus (PRRSV) emerged in the last years 48

of the decade of 1980s and by early 1990s became one of the major pathogens affecting 49

the swine industry. At present, some 20 years after its emergence (Keffaber., 1989; 50

Paton et al., 1991), PRRSV and the syndrome that it causes are still little understood 51

and fully efficacious vaccines are lacking. The reasons for such a lack of vaccines are 52

diverse but the high genetic diversity of the virus and the ability of PRRSV for 53

subverting the immune response of the host are paramount. PRRSV is a positive-sense 54

ssRNA enveloped virus classified within the genus Arterivirus. Nowadays, two 55

genotypes are recognized (I and II) that originally were described as European and 56

American because of the geographic origin of their prototypic strains (Lelystad virus 57

and VR-2332), respectively (Collins et al., 1992; Wensvoort et al., 1991). Genetic 58

diversity within genotype-I PRRSV isolates is high (Forsberg et al., 2002; Mateu et al., 59

2003; Stadejek et al., 2002) and at least three or four subtypes are being recognized 60

(Stadejek et al., 2002, 2006, 2008). 61

62

PRRSV genome is organized in 9 open reading frames (ORF). ORF1a and 1b encode 63

the viral replicase as well as other non structural proteins (nsp) (Snijder and 64

Meulenberg., 1998).Of these nsp, nsp2 is the largest and has been shown to contain a 65

large cluster of B-epitopes (Oleksiewicz et al., 2001). Recently, nsp2 and nsp1 of 66

genotype II strains have been reported to be involved in the interplay between the virus 67

and the host response (Beura et al., 2010; Chen et al., 2010, Subramaniam et al., 2010) 68

by down-regulating mediation of interferons and other cytokines. ORFs 2a, 2b and 3-7 69

encode for the viral structural proteins. Most of the abovementioned studies about 70

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genetic diversity of PRRSV have been developed by examining ORF5 or ORF7 but also 71

some papers focused on ORF3 which is thought to be a molecular clock for PRRSV 72

evolution (Forsberg et al., 2001). Surprisingly, a GenBank search for full length genome 73

–or almost complete genome- PRRSV sequences of genotype I revealed the scarcity of 74

these data and less than 10 full sequences could be found. The aim of the present study 75

was to produce and analyze the sequence of ORFs 1a to 7 of contemporary PRRSV 76

field strains of genotype-I for determining within genome diversity and phylogenetic 77

relatedness and to examine some of the immunobiological properties associated with 78

those strains. 79

80

Material and methods 81

. Strains and sequencing. Six field PRRSV strains (2982, 3249, 3256, 3262, 3266 and, 82

3267) were randomly selected among non epidemiological related strains. Additionally, 83

the vaccine strain Porcilis PRRS was also sequenced. Origin and characteristics of these 84

strains are shown in Table 1. PRRSV strains were grown in porcine alveolar 85

macrophages (PAM) obtained from high health pigs of a farm historically free from all 86

major pig diseases including pseudorabies, classical swine fever, PRRS and influenza. 87

For assessing purity of the viral production, PAM batches were tested by reverse 88

transcription (RT)-PCR or PCR for PRRSV, hepatitis E virus, Torque tenovirus and 89

porcine circovirus type 2 according to previously published protocols (Martin et al., 90

2007; Mateu et al., 2003; Quintana et al., 2002; Segalés et al., 2009). Viral batches were 91

tested for all those pathogens as well. All viral isolates were used as PAM supernatants 92

(passage n=3). Viral RNA was extracted from supernatants by using the QiaAMP viral 93

RNA minikit (Qiagen, Barcelona, Spain) according to the manufacturer instructions. 94

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Reverse transcription was done using Superscript II Reverse transcriptase and random 95

hexamers (Invitrogen, Barcelona, Spain). The obtained cDNA was then used in specific 96

PCRs designed to amplify overlapping segments of the viral genome (Table 2). Both 97

strands of PCR products were sequenced using the Genetic Analyzer 3130 XL (Applied 98

Biosystems). When deletions in the genome were found, confirmation was carried out 99

by re-sequencing of the products and synthesizing of new primers flanking the supposed 100

deletion and further amplification and sequencing. 101

. Analysis of viral sequences. Sequences were examined and purged of errors using 102

Chromas Pro 1 (Larkin et al., 2007). Predicted amino acid sequences were obtained by 103

translation using BioEdit (Hall., 1999). Nucleotide sequences were aligned using 104

Clustal X2 with correction for multiple substitutions. Bootstrap values were calculated 105

after 1000 iterations using the Neighbor-joining method. For comparative purposes 106

available full length sequences of genotype I (Genbank Accession numbers: M96262-107

Lelystad virus; AY366525-North American EuroPRRSV; AY588319-LV-4.2.1; 108

DQ864705-strain 01CB1 from Thailand; DQ489311-viral clone; EU076704-strain 109

HKEU16 from Hong Kong; FJ349261- strain KNU07 from Korea and GQ461593-110

strain SHE from China) and genotype II (Genbank accession numbers: AF066183, 111

AF325691, AY032626, AY424271, AY545985, G EU880437, EU708726, Q857656, 112

NC_001961, U87392) PRRSV sequences were also included. Synonymous (dS) and 113

non-synonymous (dNS) substitutions in each ORF were calculated using SNAP 114

(Körber., 2000) available at http://hcv.lanl.gov/content/sequence/SNAP/SNAP.html and 115

expressed as dS-dNS values. Topological trees were built up with Mega4 (Tamura et 116

al., 2007) and rooted on mid-points. Predicted amino acid sequences were also 117

examined for variations in the linear B-epitopes reported by Oleksiewicz et al. (2000, 118

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2001), and in the neutralization epitopes (NE) known to be located in GP4 (Meulenberg 119

et al., 1997) and GP5 (Plagemann., 2004). 120

. Cytokine induction in peripheral blood CD172a+ cells and PAM. Recent papers 121

indicated the role of non structural proteins in the regulation of the immune response to 122

PRRSV through cytokine release. Since full ORF1a and 1b sequences were available, 123

cytokine induction by the different PRRSV isolates was examined using peripheral 124

blood CD172a+ cells or alveolar macrophages. PBMC and PAMs were obtained from 125

healthy pigs of the same litters than above. CD172a+ cells were purified from PBMC by 126

positive selection using MACS Microbeads (Miltenyi Biotech SL, Spain). Briefly, cells 127

were incubated with mouse anti porcine CD172a-FITC (Serotec, Spain) on ice for 30 128

minutes. After incubation, PBMC were washed and CD172a+ cells were coupled (15 129

min on ice) with anti-FITC magnetic particles (Miltenyi Biotech SL, Spain). PBMC 130

were washed again, re-suspended in MACS buffer (PBS plus foetal calf serum) and 131

labeled cells were retrieved using LS selection columns (Miltenyi Biotec SL, Spain) 132

according to manufacturer’s instructions. Purity of the cellular suspension was 133

examined by flow cytometry analysis before further characterization. The obtained cell 134

suspension always had a richness of CD172a+ 92%. PAM were obtained by 135

bronchoalveolar lavage of lungs of piglets. Briefly, after humane euthanasia, lungs were 136

removed aseptically and washed by infusion through the trachea of PBS (Sigma, Spain) 137

supplemented with 2% gentamicin. The retrieved cell suspension was centrifuged (10 138

minutes at 450 g), washed and cells were frozen at -150°C until needed. PAM were 139

produced by adhesion to plastic of the retrieved cells. Parallel cultures of PAM were 140

always examined for absence of pathogens as described above. PAM and CD172a+ 141

cells were seeded at a density of 5x105 cells/well in 0.5 ml volume and stimulated with 142

viable PRRSV (2982; 3249; 3256; 3262; 3266 and 3267 strains) at 0.01 m.o.i for 24 h. 143

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All strains were examined three times (separate days), in triplicate cultures each time. 144

For a given series of tests, all strains were tested in cells coming from the same animals. 145

As a negative control, supernatants from mock-infected PAM were included. Each time, 146

cell culture supernatants of the three replicas were collected and mixed and the resulting 147

mixtures were examined by ELISA for determining concentrations of different 148

cytokines. Because of the greater sensitivity of CD172a+ for producing IFN-α, IL-10 149

and TNF-α (Gimeno et al., in press), these cytokines were examined in cell culture 150

supernatants of CD172a+ cells while supernatants of PAM were used to examine IL-1, 151

IL-6 and IL-8. IFN- capture ELISA was performed as reported previously (Guzylack-152

Piriou et al., 2004) using K9 and F17 monoclonal antibodies (Grupo Taper SA, Madrid, 153

Spain). F17 was biotinylated (Phase Biotinylation Kit, PIERCE, Spain). IFN- 154

recombinated protein (PBL Biomedical lab, Piscataway, New Jersey) was used as a 155

standard. IL-10 capture ELISA was performed using commercial pairs of mAbs (swine 156

IL-10, Invitrogen, Barcelona, Spain) (Díaz et al., 2005). TNF-, IL-1, IL-6 and IL-8 157

capture ELISAs were performed using matched antibody pairs according to 158

manufacturer’s instructions (R&D systems, Spain). The cut-off point of each ELISA 159

was calculated as the mean optical density of negative controls plus three standard 160

deviations. Values for the cytokine concentration in cell culture supernatants were 161

calculated as a corrected concentration resulting of the subtraction of cytokine levels in 162

mock-stimulated cultures from the values obtained for virus-stimulated cultures 163

(concentration PRRSV-concentration mock). 164

.Statistics. Statistical analysis was done using StatsDirect 2.7.5. 2 test was used to 165

compare proportion of substitutions in the different viral proteins; comparison of results 166

obtained in VNT and ELISPOT was done using the Kruskal-Wallis test. 167

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168

Results 169

. Alignment and comparison of PRRSV genomic and predicted amino acid (aa) 170

sequences. Global results for the comparison of ORFs and predicted proteins are 171

shown in Table 3. Average similarity of the different ORFs among genotype I strains 172

ranged from 94.7%2.4% for ORF7 to 89.5%4.4% for ORF1a. However this value for 173

ORF1a was strongly biased by the different values of similarity in each of the ORF1a-174

encoded nsp. Thus, the higher variation was found in nsp2 which average similarity 175

among examined genotype I strains (aa) was only of 83.3%6.7% followed by nsp1 176

with a 88.4%4.5%. Interestingly, most of the variation in nsp1 accumulated in nsp1 177

where between aa positions 180 and 305 up to 66 substitutions were found (53% of the 178

positions). Figure 1 shows the aa alignment for nsp1. In nsp2, variation was even higher 179

and concentrated between aa positions 269 and 430 with up to 88 positions with 180

substitutions (55%). However, the most relevant finding was the existence of deletions 181

of different length. Strains 2982 and 3262 had a 74 aa deletion (positions 308-381), 182

longer than any previously known nsp2 deletion in genotype I strains, for example 183

AY366525, DQ489311 or FJ349261 (Figure 2). Strain 3256 had a 25 aa deletion 184

distributed in two non-contiguous segments (346-358 and 418-429) and strain 3249 has 185

a 1 aa deletion (aa 349). Porcilis PRRS also had a 74 aa deletion starting from position 186

281. When nsp2 protein sequences were compared among genotype I and II PRRSV 187

strains it was found that the percentage of similarity was the lowest of all viral proteins 188

(26.3%). Similarities below 50% in the aa sequence were also found for nsp7 and 189

nsp12. Average similarities for other proteins encoded by ORF1a or ORF1b were well 190

above 90%. 191

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Examination of the other ORFs and of the corresponding predicted proteins showed that 192

ORF3 and ORF4 were the next genes with a higher variability (89.9% of average 193

similarity in both cases); however, the percentage of positions with amino acid 194

substitutions showed a trend for being higher in GP4 (57 out of 183 aa, 31%) compared 195

to GP3 (63 out of 265 aa, 24%; p=0.08). Figures 3 and 4 show the alignments for GP3 196

and GP4, respectively. For GP3 a two aa deletion was found in strain 3262 (positions 197

235-236) corresponding to the same region previously reported to be deleted for Hong 198

Kong strain EU076704. In GP4, a two aa deletion was also found in strain 3262 199

(positions 59-60) upstream of a known eight aa deletion in strain EU076704 (positions 200

57-64). Regarding GP5, it is worth noting that 30% of the aa positions could harbor a 201

substitution being the fourth viral protein after nsp2, nsp1 and GP4 in the proportion of 202

variable positions. The global analysis of dS-dNS substitutions showed that density of 203

dNS mutations was higher in nsp1, nsp2 , the carboxy-terminal segment of GP3, the 204

whole GP4 and the amino-terminal part of GP5 (Figure 5). 205

. Phylogenetic grouping of PRRSV strains. Phylogenetic analysis of whole ORFs 1-7 206

is shown in figure 6. According to the bootstrapped phylogenetic tree, strains 2982 and 207

3262 formed a cluster; strain 3266 clustered with DQ864705; 3267 clustered with 208

GQ461593 and strains 3249 and 3256 grouped together. When phylogenetic trees were 209

calculated based on the particular ORF sequences of those same strains, it was observed 210

that grouping of sequences varied depending on the ORF or even the segment used. For 211

example, in ORF4 strain 2982 formed a significant cluster with strain GQ461593 and 212

strain 3267; in contrast to ORFs 1a and 1b where 2982 clustered with 3262. In ORF5 213

strain 3249 clustered with FJ349261 but did not in any other ORF, and 3249 clustered 214

with 3267 in nsp5 (not shown). Globally, grouping obtained with ORF1b was the most 215

similar to that obtained with the whole virus sequence. In contrast, grouping based on 216

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ORF5 or ORF7 produce very dissimilar trees when compared with the one obtained 217

with the almost whole viral sequence (Figure 6). Since sequencing of whole ORF1b is 218

not useful for practical purposes, trees based on individual nsp were examined. Of 219

these, nsp9 results were the most similar to the whole virus examination (Figure 6). 220

. Variation in linear B-epitopes of PRRSV. Predicted amino acid sequences were 221

examined for changes in B-epitopes reported before in the literature. Regarding the 222

known linear epitopes described by Oleksiewicz et al. (2001) it is worth to note that out 223

of nine serologically confirmed epitopes, seven (ES1, ES3, ES4, ES6, ES7, ES9, ES11) 224

had substantial variations. Particularly, ES3 and ES4 were deleted in strains 2982 and 225

3262. Figures 1 and 2 show variations in the cluster of epitopes present in nsp1 and 226

nsp2. Regarding the epitope reported by Oleksiewicz et al. (2000) in GP3 (which core 227

was RKASLSTS), that epitope was absent in the examined sequences. Variations in the 228

core of the NE in GP4 (aa 59-66 in Lelystad virus) were found in most of the sequences; 229

deletions were even found in strains 3262 and EU076704 (Figure 4). In contrast, for the 230

NE in GP5, variations were only found regarding the glycosylation site at N-37 and 231

thus, strains 2982, 3256 and 3262 were predicted to have this third glycosylation site at 232

N-37 (not shown). 233

. Cytokine induction by different strains. Table 4 shows the results of the cytokine 234

ELISAs. All tested strains were unable to induce IFN- or IL-6 release; in contrast, all 235

of them induced very high levels of IL-8 and IL-1 and, particularly, strain 3256 induced 236

the highest IL-1 levels. Regarding TNF-, only strains 2982, 3249, 3256 and, 3262 237

induced release of this cytokine, being 3262 the strongest inducer. Strains 2982, 3256 238

and 3262 induced secretion of IL-10, being the 3256 the one yielding the higher 239

response. 240

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Discussion 241

A review of the literature about the genetic diversity and variations of genotype-I 242

PRRSV reveals that most of the papers deal with variations in ORF5/GP5 and ORF7/N 243

protein. The scarce remaining papers that examined the whole viral genome or almost 244

the whole viral genome (Amonsin et al., 2009; Fang et al., 2004; Nam et al., 2009; 245

Roop et al., 2004) were mostly focused in a particular strain or a very narrow set of 246

strains. Therefore, knowledge in that area is limited and often this has leaded to assume 247

that most facts described for a single genotype-I or genotype-II isolate could represent 248

all PRRSV strains. 249

250

The present study further confirms that diversity of genotype-I isolates of PRRSV is 251

high and, that variability within the viral genome is unequally distributed. Thus, ORF1a, 252

ORF3 and ORF4 account for the highest diversity (10.5%; 10.1% and 10.1%, of 253

variation, respectively). The examination of the topological distribution of dS-dNS 254

offers a finer picture of this. Within ORF1a, most substitutions and deletions 255

accumulate in nsp1 and nsp2 while for other nsp in that ORF1a, similarities are over 256

95%. Within nsp1, the highest variability is observed in nsp1. Recently, Beura et al. 257

(2010) showed the importance of nsp1 for inhibiting activation of the IFN- promoter 258

and that nsp1 inhibited NF- signalling. According to those authors, IL-8 mRNA 259

induction upon dsRNA treatment (mediated by TLR-3) was reduced by nsp1 260

expression. In our study, in spite of the extremely high variability found in nsp1 (up to 261

98 positions with substitutions out of 219 aa; 45%) none of the six tested strains had the 262

ability to induce detectable levels of IFN- and, contrarily to what could be expected 263

from the results of Beura et al. (2010), all strains were extremely able inducers of IL-8, 264

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suggesting either that our system (PAM) is not compatible to HEK-293 cells used by 265

Beura et al., that other parts of the virus may also be good IL-8 inducers, or that levels 266

of nsp1 necessary to produce such an inhibition of IL-8 mRNA are attainable in nsp1 267

-transfected HEK-293 cells but not in infected PAM. 268

Nevertheless, the most variable part of the virus was nsp2. In the present study the 269

length of the predicted protein varied from 861 aa (i.e. Lelystad virus) to 787 aa in 270

strains 2982 and 3262. The 74 aa deletion in nsp2 of those two isolates is the largest 271

deletion known up to date in genotype-I strains (Nam et al., 2009; Roop et al., 2004) 272

and confirm the non-essential nature of the segment comprised between residues 280 273

and 380 of genotype-I nsp2. Moreover, it is interesting to note that the most negative 274

dS-dNS values were located in nsp2 and that similarity between genotype-I nsp2 and 275

genotype-II nsp2 can be as low as 24%. These facts evidence that evolutive pressures 276

act with high intensity upon this protein and are compatible with an important role of 277

this protein in the immune regulation. Chen et al. (2010) suggested recently that 278

deletion of some epitopes in nsp2 may lead to substantial changes in the biological 279

properties of a given PRRSV strain. According to those authors, deletion of epitope ES3 280

in nsp2 probably result in an enhanced in vitro an in vivo replicative ability as well as 281

decreased ability to induce IL-1 and TNF- responses in peripheral blood 282

mononuclear cells or macrophages. In the case of the present study, strains 2982 and 283

3262 that had natural deletion of ES3 did not replicate better than strains harbouring a 284

full nsp2 (i.e. 3266 or 3267) even they yielded lower titres. However, since parental –285

undeleted- ancestor of strains 2982 or 3262 were unknown, it cannot be ascertained if 286

the deletion had or not an essential effect on the replicative ability of PRRSV in 287

macrophages but it is interesting to remark that the vaccine strain Porcilis PRRS, that is 288

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known for replicating poorly in macrophages, also harboured a 74 aa deletion in that 289

segment of nsp2. 290

Regarding the cytokine induction by the examined strains, it is worth to remark that 291

strains with extensive 25 aa or 74 aa deletions in nsp2 were the only inducing 292

simultaneously IL-10 and TNF- release. In the case of 3262 (with a 74 aa deletion 293

affecting ES3 and ES4), levels of TNF- were the highest of the six examined strains. 294

These results are in apparent contradiction with those reported by Chen et al. (2010) 295

who observed that deletion in ES3 seemed to impaired TNF- release. As seen in the 296

present paper, field strains are in fact diverse and may harbour different deletions and 297

substantial variations in nsp2. Other authors have also reported that nsp1 suppresses 298

tumor necrosis factor-alpha promoter activation (Subramaniam et al., 2010). In this 299

scenario, the biological properties of a given strain will be the result of the combination 300

of all those elements of variation and not only of a given one, and thus one may 301

counteract the effect of another. This may well explain this apparent contradiction. 302

Besides this, when strains are phylogenetically examined, it can be observed that the 303

same isolates can cluster differently when different viral proteins are looked upon. In 304

our opinion, these results were highly suggestive of differential selective pressures in 305

different genes, potential recombination or reassortment. At this point we are unable to 306

clearly determine the cause. The functional role of the specific nsps, such as nsp1α/β, 307

nsp2, nsp4, nsp7, and nsp11, is the modulation of host immune responses to PRRSV 308

infection and the nsp3–8 region has been identified as containing major virulence 309

factors (Fanga and Snijderb, 2010). In consequence, it is not uncommon observing the 310

highest genetic diversity in those target viral regions suffering direct pressure of the host 311

defence mechanisms. Interestingly, nsp9 seems to represent the whole virus better than 312

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any other else protein. As nsp9 encodes for a virus-encoded RNA-dependent RNA 313

polymerase and it is responsible of the replication of single-stranded RNA viruses with 314

genomes of positive polarity, this protein seems to be the most conserved among strains. 315

The abovementioned variability most probably will also affect antigenic properties of 316

the virus, such as the induction of NA by GP5 (Plagemann et al., 2002) and recently by 317

GP4 (Vanhee et al., 2010). When the predicted viral proteins were analyzed for the 318

variation in known linear B-epitopes, it could be seen that in the set of linear epitopes 319

reported by Oleksiewicz et al. (2001) only ES2 and ES5 were relatively conserved and 320

ES3 and ES4 can be non existent in field strains. This result suggests that the possibility 321

of constructing a marked differential vaccine by deleting some of the linear epitopes of 322

the nsp2 cluster is at least, extremely difficult. On the other hand, we have made an 323

approach considering linear B-epitopes but to have a full image relating antigenicity 324

with sequence conservation, the tertiary protein structure also matters. It could be 325

possible that tertiary structure-rich regions could interfere with the presentation of 326

strictly linear epitopes to porcine B cells and predominate for instance in functional 327

protein domains. Moreover, it is difficult to predict whether or not amino acid changes 328

would correspond with changes in the recognition by monoclonal antibodies of these 329

linear epitopes. Thus, prior to diagnostic exploitation, more information is needed about 330

the biological significance of the abovementioned changes as well as about the 331

interindividual variability in anti-ES responses. 332

In summary, taking together, the results of the present study reinforce the notion 333

(Stadejek et al., 2002, 2007, 2008) that genetic diversity in PRRSV is very high and 334

shows that nsp1, nsp2 and ORFs 3 and 4 suffer the highest degree of variation within 335

the viral genome. This variability also affects most of the known linear B-epitopes of 336

the virus and may have also an impact on the biological properties of the virus. Also, 337

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the present paper shows that different strains can have substantially different 338

immunobiological properties. These data can contribute to the understanding of the 339

complexity of PRRSV. 340

341

Aknowledgements 342

Our grateful thanks to Núria Navarro, Esmeralda Cano, Anna Coll and Montse Tello for 343

their assistance in the laboratory. We would also like Dr. María Montoya for her help in 344

obtaining CD172a+ cells and Dr. Martí Cortey for his helpful assistance in the genetic 345

analysis. This project was supported by a grant from the Spanish Ministry for Science 346

and Innovation (MICINN) reference AGL2008-05708-C02-02/GAN. Ivan Diaz was 347

supported by the project CSD-007-Porcivir of the program Consolider Ingenio 2010. 348

349

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References 350

1. Amonsin A., Kedkovid R., Puranaveja S., Wongyanin P., Suradhat S., 351

Thanawongnuwech R., 2009. Comparative analysis of complete nucleotide 352

sequence of porcine reproductive and respiratory syndrome virus (PRRSV) 353

isolates in Thailand (US and EU genotypes). Virol. J. 6,143. 354

355

2. Beura L.K., Sarkar S.N., Kwon B., Subramaniam S., Jones C., Pattnaik A.K., 356

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Table 1. Characteristics of PRRSV strains used in the present study.

Strain reference

Country Year of

isolation Clinical signs

(field) Tissue

Maximum titer in PAM* (TCID50/ml)

2982 Spain 2005 Respiratory (weaners)

Lung (weaner)

104.7

3249 Spain 1992 Abortions Serum (sow)

104.7

3256 Spain 2005 Respiratory (weaners)

Lung (weaner)

104.7

3262 Spain 2005 Respiratory (weaners)

Lung (weaner)

105.9

3266 Germany 1992 Abortions Serum (sow)

107.5

3267 Portugal 2006 None (boar)

Serum (boar)

107.3

*PAM = Porcine alveolar macrophages

TCID50 = Tissue culture infectious dose 50%

Table 1

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Table 2. List of oligonucleotide primers utilized for amplifying the ORFs of PRRSV strains.

Fragment Primer pair Primer sequence a

(5’to 3’ sense) Annealing

Tª (°C)

Primer

location b

ORF 1 1.0F CCTGTTCTAGCCCAACAGGT 55 93

1.0R AGCCGCGCTCGTAAACT 55

1.1F TGCTGATGTTTTGTACCGTGA 55 524

1.1R CAGCACTTGCCATCAAACAC 55

1.1B-Fc

TTCCTCCAACGACTTGTGARGGTTGC 55 501

1.1B-Rc

CCATGCACGCCCCACTTGGTTTG 55

1.2F GTGTTTGATGGCAAGTGCTG 55 1032

1.2R ATTATGGCGGCAAGGACAT 55

1.3F GACAGACGGGTCTTGTGGTT 55 1493

1.3R CATTTTTGCGGAACAACCTC 55

1.4F GAGGTTGTTCCGCAAAAATG 55 2007

1.4R CTGGTTGGGACAAATCCAGT 55

1.4B-Fd,e

TGTCCGAGYTCYAAACAGGCCATG 51 2079

1.4B-Rd,e

CGGCATCAGAACCTGGGTTGT 51

1.5F ACGAACCACTGGATTTGTCC 55 2518

1.5R GAGTTCGGCAAATTCGAAAG 55

1.6F TTCTCCGCACAAGCCTTAAT 55 3021

1.6R CTGATAGACCCCGCGAGAC 55

1.6B-Fd,e

CCTGGACCAGCCTTTAGATCT 50 2729

1.6B-Rd,e

CTGGGAGATGGGAGACAATTTC 50

1.7F GGGCGGGAGTTGGAAA 55 3488

1.7R CGTGACCCACCGAGTAACTT 55

1.8F CGGTGGGTCACGTTATCTCT 55 3951

1.8R AGCCACAAAAGTGTCCGAAT 55

1.9F TGGACCAGCCTACACCTGA 55 4378

1.9R TGGGTCAGCGGTTCCTC 55

1.10F GACCCATGGTGTTCAAATCC 55 4716

1.10R TGTAATAATTCCGGCAACCTG 55

1.11F TCTCAGCCGTGGCACA 55 4819

1.11R CGGTGTTAAGGCAGGGTTT 55

1.12F CCTGCCTTAACACCGTGAAT 55 5323

1.12R CCAAGTCACTCGGAATGGAT 55

1.13F CCTAGCCTCCGTCCCTGTA 55 5885

1.13R TAGGAAGAAGGCGCTTGAAA 55

1.13B-Fd

CGAGCGTTCCCCTTGGAGACAT 55 5883

1.13B-Rd

CAGCCGTTAGGGACTCGTTACTCA 55

1.14F TTTCAAGCGCCTTCTTCCTA 55 6385

1.14R GCGGACCATTCTCAAAAAGA 55

1.14B-Fc,f

GTGGCTATTCAAATACCGGTGC 45 6332

1.14B-Rc,f

TTCATCCTCTCAACTTTGAGATC 45

1.15F CCCCTCTTTTTGAGAATGGT 55 6901

1.15R AAAGCCTGTTCAGTGGTCAA 55

1.16F TTGACCACTGAACAGGCTTT 55 7383

1.16R GATTGCGGACTTGGTGTCTT 55

1.17F CAAGACACCAAGTCCGCAAT 55 7916

1.17R GGTTTTGGGCTTGGAACAGT 55

1.17B-Fc

TGTTAGGGGGGATCCTGAGC 52 7840

1.17B-Rc

CCAAGCCTTCTTCATGAATGCCTG 52

1.18F GTTCCAAGCCCAAAACCAG 55 8400

1.18R CTCGACCCACCAGTGGTAAT 55

1.19F ATTACCACTGGTGGGTCGAG 55 8964

1.19R GGTGACTGACACTGCGAACA 55

1.20F CGCAGTGTCAGTCACCTGTT 55 9480

1.20R CACACAAGGGGTGTTTTGG 55

1.21F CCAAAACACCCCTTGTGTGT 55 10047

1.21R CGAAATCGAGATGGACCTGT 55

1.21B-Fe

GTGTCGTACCTCGATGAGGC 55 9989

1.21B-Re

GCCCGAGTGATGGCTACAAG 55

1.21-22F CTTGTAGCCATCACTCGGGC 50 10400

1.21-22R CCCAGATTATGTGCCACTTGCG 50

1.22F CTCTCTTAGCCGCTTGTTCG 55 10623

1.22R GAGTCCTTAGGCAGGGACCTA 55

1.23F CCTACCTAGGTCCCTGCCTAA 55 11107

1.23R GATTGCCCAATCCTCAGTGT 55

1.24F CAATCCTTGCACGCCGTAT 55 11604

Table 2

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a Primers were initially designed based on sequence information available for LV (genBank accession number

M96262); b Numbers correspond to position within the LV genome; Tª, temperature; L, left or forward primer;

R, right or reverse primer; c

primers used for amplifying 3256 strain; d

primers used for 3262 strain; e

primers

used for 2982 strain; f primers used for 3249 strain

g Díaz et al. 2006 (Virology 351: 249–259).

1.24R CAAGTGGGAAACTCGCATGT 55

1.24B-Fc

ACCTGGCAGTGACACCGTATGA 52 11463

1.24B-Rc

GAAAGCGTCCACGAACAGCTGGC 52

ORF2 2F g CTGGCACAGAATTGCAGGTA 55 11724

2R g GCACACTGATGAGCCATTGT 55

2-3F TGCTCCGCGCTTCTCCGTTCG 50 11951

2-3R ACATAGCGTAGAGCTGGAATTCG 50

ORF3 3Fg ACAATGGCTCATCAGTGTGC 55 12401

3R g TGAAGCCTTTCTCGCTCATT 55

ORF4 4Fg AGCGTGACCATGATGAGTTG 55 12654

4R g AAAAGCCACCAGAAGCAAGA 55

4B-Fd

GACGGGGGYAATTGGTTYCA 47 12920

4B-Rd

CGCCCCAATTTSTGAGAACATCTC 47

ORF5 L1F g

TGAGGTGGGCTACAACCATT 55 13445

L1Rg AGGCTAGCACGAGCTTTTGT 55

ORF6 6F g GTCCTCGAAGGGGTTAAAGC 55 14037

6R g CTGTCCTCCCCTAGGTTGCT 55

ORF7 7Fg GGCAAACGAGCTGTTAAACG 55 14555

7R g AATTTCGGTCACATGGTTCC 55

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Table 3. Similarity of the examined ORFs and of the predicted amino acid products of the 6 field strains and 9 European type PRRSV from the GenBank.

ORF/Protein Length (aa) Similarity gen. I*

(mean sd)

Range Substitutions

(nº aa; percentage¶)

Similarity gen. II**

(mean sd)

Range

ORF1a NA 89.5%4.4% 99.8-82.3% ND 52.2%0.4% 52.9-51.3%

Nsp1 385 aa 88.4%4.5% 100-82.0% 132; 34.3% 52.2%0.9% 54.3-50.5%

Nsp2 861, 844, 841, 836, 787 83.3%6.7% 99.7-70.6% 414; ≥48.1% 26.3%0.8% 28.0-24.4%

Nsp3 447 95.2%2.2% 100-89.5% 77; 17.2% 56.7%0.6% 58.0-55.3%

Nsp4 203 95.0%4.3% 100-90.1% 39; 19.2% 60.5%0.8% 62.2-58.8%

Nsp5 170 95.5%2.3% 100-91.1% 28; 16.4% 71.0%1.7% 73.5-66.4%

Nsp6 16 95.3%4.4% 100-81.2% 3; 18.8% 78.3%3.1% 81.2-75.0%

Nsp7 270 96.3%1.5% 100-92.9% 39; 14.4% 44.6%0.8% 46.5-43.2%

Nsp8 44 97.5%2.5% 100-90.9% 5; 11.4% 67.1%2.0% 70.4-61.3%

ORF1b NA 93.2%3.1% 99.8-87.3% ND 62.7%0.3% 63.3-62.0%

Nsp9 645 97.9%1.0% 100-96.1% 48; 7.4% 74.0%0.4% 75.0-73.2%

Nsp10 442 96.0%2.2% 100-90.4% 77; 17.4% 63.4%0.5% 64.7-62.2%

Nsp11 224 97.4%1.2% 100-94.6% 26; 11.6% 74.7%1.0% 77.4-72.1%

Nsp12 152 95.0%2.9% 100-88.1% 26; 17.1% 39.3%0.6% 40.7-38.2%

ORF2a NA 93.2%3.0% 99.4-86.9% ND 63.8%0.7% 66.9-62.6%

GP2a 249 93.3%3.0% 98.7-87.5% 61; 24.4% 60.9%1.1% 65.2-57.8%

P2b 70 96.4%3.3% 100-88.5% 9; 12.8% 69.5%2.0% 75.3-64.3%

ORF3 NA 89.9%5.2% 100-80.5% ND 62.1%0.7% 63.7-60.6%

GP3 265, 263, 257 87.5%6.3% 100-77.3% 63; ≥23.7% 54.9%1.2% 57.3-54.2%

ORF4 NA 89.9%5.0% 100-80.9% ND 64.5%1.2% 66.8-77.0%

GP4 183, 181, 175 88.5%5.7% 100-77.0% 57; ≥31.1% 66.8%2.3% 70.4-59.5%

ORF5 NA 91.1%3.7% 99.8-84.0% ND 60.7%0.7% 62.4-58.5%

GP5 202 90.0%3.5% 99.5-84.0% 62; 30.7% 54.7%1.3% 56.5-50.7%

ORF6 NA 93.8%3.3% 100-88.1% ND 69.2%0.7% 69.2-71.2%

M 173 94.6%2.8% 100-89.0% 32; 18.5% 78.8%1.2% 81.6-75.2%

ORF7 NA 94.7%2.4% 100-89.4% ND 61.7%0.7% 63.0-60.0%

N 127 95.0%3.3% 100-88.2% 24; 18.9% 58.6%1.1% 61.0-55.7%

* Similarity gen. I: Similarity among genotype I strains; ** Similarity gen. II: Similarity of genotype I with genotype II strains; ¶ Percentage was calculated over

the longest aa length. NA; not applicable. ND; not done.

Table 3

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Table 4. Cytokine levels in cultures of peripheral blood CD172a+ cells (IFN-, TNF- and IL-10)

or porcine alveolar macrophages (PAM) (IL-1, IL-6 and IL-8) stimulated with different viable

PRRSV isolates at 0.01 multiplicity of infection as determined by capture ELISA. Values are

shown in pg/ml.

*Medium-stimulated cells.

Cytokine levels in cell culture supernatants (pg/ml)

Strain IFN-α TNF-α IL-1 IL-8 IL-6 IL-10

2982 Neg. 376 304 >8000 Neg. 212

3249 Neg. 103 216 >8000 Neg. Neg.

3256 Neg. 437 1232 >8000 Neg. 723

3262 Neg. 1119 411 >8000 Neg. 461

3266 Neg. Neg. 301 >8000 Neg. Neg.

3267 Neg. Neg. 302 >8000 Neg. Neg.

Mock cells* Neg. Neg. Neg. <1000 Neg. Neg.

Table 4

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Figure 1. Graphic view of the alignment of the predicted amino acid sequences of non structural protein 1 (1 and 1) for the examined PRRSv

strains. Shadowed upper box shows nsp1. Linear B-epitopes described by Oleksiewicz et al. (2001) are shown as dotted boxes with the label

ESx.

10 20 30 40 50 60 70 80 90 100

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|

M96262 MSGTFSRCMCTPAARVFWNAGQVFCTRCLSARSLLSPELQDTDLGAVGLFYKPRDKLHWKVPIGIPQVECTPSGCCWLSAVFPLARMTSGNHNFLQRLVK

2982 .P.....................Y........P..P........A.I..........P......................I..I................

3249 .......................Y...........P.....S...GI.......................S.........I...................

3256 ...................................PL....A....................V.........I.......IL.................R

3262 .P.....................Y........P..P........A.I..........P......................I..I................

3266 ....................................................................................................

3267 ........................................E...A.I......K..........................I.L................R

Porcilis ....................................................................................................

AY588319 ....................................................................................................

AY366525 .C.SV........V..............V...A..................R...............A................................

EU076704 .......................Y...........P........A.I..........R.R..........S........GI...................

FJ349261 ..................S.............P..P.....PG...I......K.....R..V.................I...................

DQ864705 ....................................................................................................

DQ489311 ................................P............V.......K..I...........................................

GQ461593 ...................................P....E...A.I......K..........................I..................R

110 120 130 140 150 160 170 180 190 200

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|

M96262 VADVLYRDGCLAPRHLRELQVYERGCNWYPITGPVPGMGLFANSMHVSDQPFPGATHVLTNSPLPQQACRQPFCPFEEAHSSVYRWKKFVVFTDSSLNGR

2982 ...........T....C.........S......................................................D....QE..I.V..PID..

3249 .........W..........IH................A.Y...........................................K.....I.....P...

3256 ............S...........................Y........................................D....E.L.I.V...PD..

3262 ...........T.Q............S......................................................D....QE..I.V..PID..

3266 ................................................................................................P...

3267 .................DH....................................................................R........P...

Porcilis ...........T....C.........S....................F.............L..................FD....QE..I.V.FPID..

AY588319 ....................................................................................................

AY366525 .................................................................................G........I.S..P...Q

EU076704 ..E.........S.........................A.Y........................................D....Q.Y.I.F..LA...

FJ349261 .................................................................................D.F.RN.....M.PPSG..

DQ864705 ................................................................................................P...

DQ489311 ..........................S.......................................R..............D........I.....P...

GQ461593 .......................................................................................R........P...

Figure 1

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210 220 230 240 250 260 270 280 290 300

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|

M96262 SRMMWTPESDDSAALEVLPPELERQVEILIRSFPAHHPVDLADWELTESPENGFSFNTSHSCGHLVQNPDVFDGKCWLSCFLGQSVEVRCHEEHLADAFG

2982 .......G.S...G..E..A..RC.............SA..TE..F.....R....SA.....Y.A..........L.....ELPT..QR...L..N...

3249 ..........G.......................S......T.........S..............R..............S...T..HRC..R......

3256 .......G.....D........G......V.........N......A....H....S......Y.A.DH.I..........F.L.AK.WR...Y..S...

3262 F......G.S...E..E..A..R..............S...TE........R....SA.....P.A..T.............ELPT..WR...L..S...

3266 ........................................................................EC..........................

3267 .......................................N...................Y...............F.........A...R........L.

Porcilis .......G.S...G..E..G.IRC.............FA..TE..F.....R.................................A..............

AY588319 ....................................................................................................

AY366525 ..I....K........E......................N.......G..................R.SN........T.................N...

EU076704 .....ALG....V..........HR................T..............S......Y.A.......S.......Y.L.TR.WR...Y......

FJ349261 R......G....TN..A............T.........N.S.......S.H....S.F....Y.A....G.N........FDL.T..WR...Y...TL.

DQ864705 ....................................................................................................

DQ489311 F............................T........IN................G.......I....N........T......A..CY......N.L.

GQ461593 .......................................N...................Y.........................A...R........L.

310 320 330 340 350 360 370 380

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|

M96262 YQTKWGVHGKYLQRRLQVRGIRAVVDPDGPIHVEALSCPQSWIRHLTLDDDVTPGFVRLTSLRIVPNTEPTTSRIFRFGAHKWYG

2982 .......P............V...........................E................S.....AF......V.....

3249 .......P............L.....S......................NS.............E.......L............

3256 .................IN........N...............................V.....S......LQV..........

3262 .......S........................................E................S.....AF......V.....

3266 ..................C..........................................I.......................

3267 .......P.......................................F..N....................A.............

Porcilis ..................H.............................N....................................

AY588319 .....................................................................................

AY366525 ......................................S.........N............I...........Q...........

EU076704 .................IN.V.............................EI.......M............LQV.....Q....

FJ349261 .................IN.V.............................E.....I..M............LQV..........

DQ864705 .......................................H.....................I.......................

DQ489311 ....................M.................S...V.....NN...........I...S.....AF............

GQ461593 .......P...............................R.......F..N....................A.............

ES1

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Figure 2. Graphic view of the alignment of the predicted amino acid sequences of non structural protein 2 for the examined PRRSv strains.

Linear B-epitopes described by Oleksiewicz et al. (2001) are shown as dotted boxes with the label ESx.

10 20 30 40 50 60 70 80 90 100

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|

M96262 AAGKRARAKRAAKSEKDSAPTPKVALPVPTCGITTYSPPTDGSCGWHVLAAIMNRMINGDFTSPLTQYNRPEDDWASDYDLVQAIQCLRLPATVVRNRAC

2982 ............R.GNG........P.....E.................................P...............A..............G...

3249 ...R.........K.EG........Q...I......................V...M.D......................A......Q....I...H..

3256 .......T.....T.....AA..ATK.T.A.ET.......................M.D......A...............I.V..H.Q...AI......

3262 ............R.GNGP.......P.....E.I.....P.........................P...............A..............G...

3266 ........................................................................................Q...........

3267 ................I........Q.....E........................M........................A......Q...........

Porcilis ........................................................................................Q...........

AY588319 ....................................................................................................

DQ864705 ............R......L....................................................................Q...........

GQ461593 ................I........Q.....E........................M........................A......Q...........

AY366525 ..........T..GG...V.AL.......A..........................M.D......................A......Q...........

DQ489311 ...........T..G....LA..I.P..........................V............P...............A......Q...........

EU076704 ..............V..LTA..D..QSI.A...A..............I...V...M..N.....................A......Q....I......

FJ349261 ...................IA..A.K.I.A.ET...................I...V.N.S....................A......Q....I..S...

110 120 130 140 150 160 170 180 190 200

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|

M96262 PNAKYLIKLNGVHWEVEVRSGMAPRSLSRECVVGVCSEGCVAPPYPADGLPKRALEALASAYRLPSDCVSSGIADFLANPPPQEFWTLDKMLTSPSPERS

2982 .....................I....................S..........................T..V.....D..........R..........

3249 .......R...........P.......P.......................E................................................

3256 ......V............................................E.......T.........C....N.........................

3262 .....................I..................A.S...................G......A..V.....D.....................

3266 .....................................................................G..............................

3267 ...................P........P...........I.....Q......................G..............................

Porcilis ...........................P........................................................................

AY588319 ....................................................................................................

DQ864705 ....................................................................................................

GQ461593 ...................P....................I.....Q......................G..............................

AY366525 .....................................................................C..............................

DQ489311 ..............................................................................D.....................

EU076704 ...........................P..............S...KN...S.................C........D...................Q.

FJ349261 ...................P......................SS..EI...E..........................D..-..................

ES2

Figure 2

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210 220 230 240 250 260 270 280 290 300

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|

M96262 GFSSLYKLLLEVVPQKCGATEGAFIYAVERMLKDCPSSKQAMALLAKIKVPSSKAPSVSLDECFPTDVLADFEPASQERPQSSGAAVVLCSPDAKEFEEA

2982 ........................V................I...K.........S...............V..T.L....GF.I...AS.L......G.

3249 ...........A............T.............E.............................P..S..T...K.......A.M.P....V..GT

3256 .................D.....LF...D...AG.....................S..........E.P..S...........D.T......NSE..A..

3262 ........................V................I...K.........S...............V..T.L....GF.I...AS.L......G.

3266 ...............N....................................................P....................R..........

3267 ........................V...........................................P..S...F.................I......

Porcilis ........................M............F...I...................G......P.......P...--------------------

AY588319 .............................................................................K......................

DQ864705 ....................................................................P...............................

GQ461593 ........................V...........................................P..S...F.................I......

AY366525 ......N...........V.....T.......M.....E..........................A..P.....T..K.........A...S..EG....

DQ489311 ........................V............PE..........................AG.P......F......P....A.......G..GT

EU076704 ........................T.............E......T......................S...R....DGT.N.....T.H...V....G.

FJ349261 S.......................T.........................................N.P..L.L.P.KK.....TV...R..G.......

310 320 330 340 350 360 370 380 390 400

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|

M96262 APEEVQESGHKAVHSALLAEGPNNEQVQVVAGEQLKLGGCGLAVGNAHEGALVSAGLINLVGGNLSPSDPMKENMLNSREDEPLDLSQPAPASTTTLVRE

2982 T......--------------------------------------------------------------------------.......S.L.A....E.K

3249 .S....GCD...I..VA....HGKK.A..I...R..FD..D...RGVG-...A..SPVD.AD.D.PF..ST....P..WGH......RS.S.A.......

3256 .LGG..G...E.I..VVP..ESDRK.A.MA..DR.E..D..ST..G-------------AQY.IQDSAG.I...TFD.WGN......RS...A.K....R

3262 T......--------------------------------------------------------------------------G......S.LVT..A.E.K

3266 ..................................................V.............S....S......................A.......

3267 .......G.............L..........A.....S.......T.G.VP...SP...AD...P...S..................S.L.T.......

Porcilis ------------------------------------------------------...............S......................A.......

AY588319 ....................................................................................................

DQ864705 ..................................................V..................S..........N...........A.......

GQ461593 .......G.............L..........A.....S.......T.G.VP...SP...AD...P...S..G..P.GW.........S.L.T.......

AY366525 ...G...R.........F.K..............Q........I..-----------------AQ..LNS.....RS..............VAA...E..

DQ489311 .S..A.........AVP..................E.......I.S-----------------AQ.S..SKR...H...........H....A.....G.

EU076704 ..M.T..GNY..D....P.G..DKK...A......EIS...S....V...V.A.S.P...IS...PSPGF...DT...WK........S..VTAM....G

FJ349261 .L.........T....PHTK...K......V.........D.V......DV.....P.D.AS-------------------......R....A...PEG.

ES3 ES4

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410 420 430 440 450 460 470 480 490 500

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|

M96262 QTPDNPGSDAGALPVTVREFVPTGPILCHVEHCGTESGDSSSPLDLSDAQTLDQPLNLSLAAWPVRATASDPGWVHGRREPVFVKPRNAFSDGDSALQFG

2982 .VL.........F.....ALA....M..R................PF...P.....D..........................I.........E.V..L.

3249 ......D.S.D.PS...QG....E..PRL..................V.R............................C........S.L.....V....

3256 .MSN..SPG.DT....N------------.G.....A..GD......N.R.PG.......T.....S.............L......GTL...G.V....

3262 .VL.........F.....ALA....MP.R................PF...P.....D..........................I.........E.V..L.

3266 ...........................R.I........................................................Q........P....

3267 .....L..G.......I......R..PR.......................P...................................G.......V....

Porcilis ...........................R.................Q......................................................

AY588319 ....................................................................................................

DQ864705 ...........................R........................................................................

GQ461593 .....L..G.......I......R..PR.......................P...................................G.......V....

AY366525 ..............A....S.....M.R...................Y........D....V...K.....................K....S...F...

DQ489311 ..........S...IA..G........R...................F........D........K.........R..C....L...K............

EU076704 L...Y....I........A..S..LRFH....R..KP....L........A.N...D...TD...KT........Y.........R.D............

FJ349261 H..E...F.....L..T.K...A.LM.H...........G..........IPG...D........KS.....................V......V....

510 520 530 540 550 560 570 580 590 600

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|

M96262 ELSESSSVIEFDRTKDAPVVDAPVDLTTSNEALSVVDPFEFAELKRPRFSAQALIDRGGPLADVHAKIKNRVYEQCLQACEPGSRATPATREWLDKMWDR

2982 ..............E.I......A..AA..K...A.....L.....................E............................D..N.....

3249 ....P..AT.......V.........M.......E.N..K..K...............................................K.........

3256 .................T.........A......E....K..........................R...............................E.

3262 ..............E.I......A...V..K...A.....L.....................E...............................N.....

3266 ...............N........N..............K............................................................

3267 .......I...................V......GI...K............................................................

Porcilis ......P.............................N..K............................................................

AY588319 ....................................................................................................

DQ864705 .....R.................................G............................................................

GQ461593 .......T...................V......GI.............................V..................................

AY366525 K....G..................GS........IA......................................R...............K.........

DQ489311 ............Q...TL.A..............A...S..V..R...H...................................................

EU076704 ...................A..............A....K.S.PR.................E......S.......L............K....R.C..

FJ349261 G...............TL.....I..........M...L...............................Q...R...............KK...R..A.

ES5

ES6

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610 620 630 640 650 660 670 680 690 700

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|

M96262 VDMKTWRCTSQFQAGRILASLKFLPDMIQDTPPPVPRKNRASDNAGLKQLVAQWDRKLSVTPPPKPVGPVLDQIVPPPTDIQQEDVTPSDGPPHAPDFPS

2982 ......................................S...E....R...........EPS.L..T.S.P..T.SQ...V...N..S....L.VLNS..

3249 ...........................................H...................L.S.E.A...TI....N..........R...S..L..

3256 ...............C...........................H...................L.S.ESA...TA.L...A.....P......R...PS.

3262 .....................Q................S...EK.C.............EPS.L..T...P..T.SQ...V...N..S....L.VLNS..

3266 .........................................................................T..........................

3267 ...............H......................S........................L.....A.G.T................R...V..L..

Porcilis .................................................P.......................T..........................

AY588319 ..................................................................................................L.

DQ864705 .........................................................................T..........................

GQ461593 ...............H......................S.............R..........L.....A.G.T................R...V..L..

AY366525 ......C......................................D.............M...Q...E.....T.S....T...................

DQ489311 ....................................K......S........R..K.........SA.L....T...........A.....LS..S..S.

EU076704 .....................................R....NK...........G...S...Q.......A.T.L..MGG............Q.L.LS.

FJ349261 .......................................H.R.S....R.............LQR.AE..P...A.S.MGV..DG....S...Q.....G

710 720 730 740 750 760 770 780 790 800

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|

M96262 RVSTGGSWKGLMLSGTRLAGSISQRLMTWVFEVFSHLPAFMLTLFSPRGSMAPGDWLFAGVVLLALLLCRSYPILGCLPLLGVFSGSLRRVRLGVFGSWM

2982 Q...SRG..D.VF....F...A..........IV..................................................................

3249 ..N......V...P....V.....H..................I.....T.............F.........VF............V............

3256 .TN......MF.F........V..H..A.....................T..S....................V.............V............

3262 Q...SRG..D.V.....F..PA..........IV..................................................................

3266 ....................................................................................................

3267 ........................H................................................V..........................

Porcilis ....................................................................................................

AY588319 ....................................................................................................

DQ864705 Q...................................................................................................

GQ461593 ........................H................................................V..........................

AY366525 .........D..C........................................................H..............................

DQ489311 ....SW...............AG..........Y......I...........................................................

EU076704 ....S.G..N..PFD...M....H......C.IV......................C.............P...F............V............

FJ349261 ..D......S..........F...........IL..............................V......................V............

ES7

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810 820 830 840 850 860

....|....|....|....|....|....|....|....|....|....|....|....|.

M96262 AFAVFLFSTPSNPVGSSCDHDSPECHAELLALEQRQLWEPVRGLVVGPSGLLCVILGKLLG

2982 .............................................F...............

3249 ...........D......N..........................................

3256 ........A..DS................................................

3262 .............................................F...............

3266 ...........................................F.................

3267 ...........D.................................................

Porcilis .............................................................

AY588319 .............................................................

DQ864705 .........................Q...................................

GQ461593 ...........D.................................................

AY366525 .............................................................

DQ489311 .............................................................

EU076704 ...........D.................................................

FJ349261 ..........TD......N..........................................

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Figure 3. Partial graphic view of the alignment of the predicted amino acid sequences of GP3 of the PRRSV strains examined. The dotted box

shows the epitope reported by Oleksiewicz et al. (2000).

210 220 230 240 250 260

....|....|....|....|....|....|....|....|....|....|....|....|....|

M96262 RSPVSPVSRRIYQILRPTRPRLPVSWSFRTSIVSDLTGSQQRKRKFPSESRPNVVKPSVLPSTSR

2982 .......F..T.R..............................GLL........ARL......L.

3249 ..........T.....L...............A...........TL..G........L.F.....

3256 ...A...F..T....KL...............A..V.RI.HL..ML.VGN....L.T.AFLT...

3262 ...A......T.....L...Q...LL..MK.TA.--.KA.KHRGSL.PK.HL.....LAF.N...

3266 .................................................................

3267 ............................................T.........AR...F...L.

Porcilis .................................................................

AY588319 .................................................................

DQ8647050 .................................................................

AY366525 ....................Q...............MR.....G....G....A....A..NI..

EU076704 .L.ANL.........................P.P---.LTGHS-----A.HL..AR..AFL....

GQ461593 ............................................T.........AR...F...L.

FJ349261 ...A..A................I.....KLN..S...A...S.A..IRNSR..A...APH....

DQ489311 .................................PG..RP....V...P.....A.....F.N...

Figure 3

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Figure 4. Graphic view of the predicted amino acid sequences of GP4. The dotted box show the location of the neutralization epitope reported

by Meulenberg et al. (1997).

10 20 30 40 50 60 70 80 90 100 110

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|

M96262 MAAATLFFLAGAQHIMVSEAFACKPCFSTHLSDIETNTTAAAGFMVLQDINCFRPHGVSAAQEKISFGKSSQCREAVGTPQYITITANVTDESYLYNADLLMLSACLFYA

2982 ....I........YL.................N.K.................L.........GTTP.R.P........................................

3249 ..T.I..L......L.................N...............N...L..Y...TT..NT....P........I...............................

3256 ..T.I.........L...................K.............N.D.L.R.PD.TPEKNAPR..PP..P.DI.I.H............-.NH.....H.......

3262 ..E.I..L..S...LV.C................K.........I..HE....HKGPE--T.GLTPTQ..P...Q.I.I.....M.........................

3266 .......L......................................................................................................

3267 ....I..L.....YL...................K.................L......T...N.....P........I...............................

Porcilis .......L.....Y......................................L..........E.P..........................................H.

AY366525 ....I..L..........................K.....................E...T.RE.P.R..........................................

AY588319 ..............................................................................................................

DQ489311 ....AF.L.V........................K.............N...S...EA..T.GQVPSR..........V...............................

DQ864705 ....A..L....................................................................................................H.

EU076704 .V..I..L.VS...LV................N.K.........L.....T.A...--------GTL..P........I...............................

FJ349261 ....I..L.V...RL...................K....P..N.....K.E.LQ..RG.TT.QSVPYK.Q....Q...................................

GQ461593 ....I..L.....YL...................K.................L......T...N.P...P........I...............................

120 130 140 150 160 170 180

....|....|....|....|....|....|....|....|....|....|....|....|....|....|...

M96262 SEMSEKGFKVIFGNVSGVVSACVNFTDYVAHVTQHTQQHHLVIDHIRLLHFLTPSAMRWATTIACLFAILLAI

2982 ................................V......................T.................

3249 .......L............................................S....................

3256 .V..D-.NE................................A..........S....................

3262 ..........................................VN.............................

3266 ......................................................................W..

3267 ................................I......................T.................

Porcilis ............W............................................................

AY366525 .......................................................T.................

AY588319 .........................................................................

DQ489311 ...........................................N.............................

DQ864705 .........................................................................

EU076704 .........................................A...............................

FJ349261 ..........V..................................V......S..T.................

GQ461593 ................................I......................T.................

NE

Figure 4

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Figure 5. Topology of the synonymous-non synonymous substitution rate in codons (ORFs1-7) of 15 European type PRRS strains. Annotation in

the lower part of the graph indicates topology of each ORF. Boxes located in the center show the location of non structural proteins in

ORF1a/1b. Y-axis show the rate of synonymous-non synonymous substitutions per codon; X-axis show the number of codons starting at nsp1.

Synonymous-Non synonymous rate

-1,2

-1

-0,8

-0,6

-0,4

-0,2

0

0,2

0,4

0,6

0,8

1

1,2

1 201 401 601 801 1001 1201 1401 1601 1801 2001 2201 2401 2601 2801 3001 3201 3401 3601 3801 4001 4201 4401 4601 4801 5001

1a 1b 2 3 4 5 6 7

nsp1 nsp2 nsp3 nsp4 nsp5 nsp7 nsp9 nsp10 nsp11

nsp6 nsp8 nsp12

Syn

on

ymo

us

No

n s

yno

nym

ou

s

Figure 5

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Figure 6. Mid-point rooted tree based on the genomic sequence of nsp9, on full ORFs1-7 sequences and

ORF5 and ORF7 PRRSV sequences. Bootstrap values are shown in the branches.

AY545985 AY424271 AF325691 AY032626 EU880437 EU708726 GQ857656 U87392 AF066183 NC001961 FJ349261 2982 3262 AY366525 DQ489311 DQ864705 3266 M96262 AY588319 Porcilis GQ461593 3267 3256 3249 EU076704

1000

1000

1000 1000

998

1000

1000

1000

1000

1000

1000

954

195

1000

862 861

861

1000

1000 1000

1000 997

AY032626 EU880437 EU708726 GQ857656 AF325691 AY545985 AY424271 U87392 AF066183 NC001961 3262 EU076704 FJ349261 3249 DQ864705 3266 M96262 AY588319 Porcilis 2982 AY366525 DQ489311 GQ461593 3267 3256

747

993

1000

432

1000 278

913

1000

1000

1000

392

155

1000

473

312 967

842

635 984

907

773 997

GQ857656 EU880437 EU708726 AY032626 U87392 AF066183 NC001961 AF325691 AY545985 AY424271 2982 EU076704 FJ349261 M96262 AY588319 Porcilis DQ864705 DQ489311 AY366525 GQ461593 3267 3266 3249 3256 3262

275

591 1000 691

844 1000

742

491

1000

1000

409

479

713 988

676

256

414

644 488

943 541

723

ORF7

Full ORF1-7

Nsp9

ORF5

AY545985

AY032626

AY424271

EU880437

EU708726

GQ857656

AF325691

U87392

AF066183

NC - 001961

2982

3262

3266

Porcilis

M96262

AY588319

DQ864705

AY366525

DQ489311

GQ461593

3267

3256

3249

FJ349261

EU076704

965

186 528

551

537

765

523

991

993

1000

685

493

981

641

902

480 960

674

500 724

551

661

Figure 6