Genomic sequence and virulence comparison of four Type 2 porcine reproductive and respiratory syndrome virus strains

Virus Research 169 (2012) 212–221

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Genomic sequence and virulence comparison of four Type 2 porcine reproductive and respiratory syndrome virus strains Susan L. Brockmeier a,∗ , Crystal L. Loving a , Ann C. Vorwald a , Marcus E. Kehrli Jr. a , Rodney B. Baker b , Tracy L. Nicholson a , Kelly M. Lager a , Laura C. Miller a , Kay S. Faaberg a a b

Virus and Prion Research Unit, National Animal Disease Center, USDA, Agricultural Research Service, Ames, IA, United States Veterinary Diagnostic and Production Animal Medicine, Iowa State University, Ames, IA, United States

a r t i c l e

i n f o

Article history: Received 20 March 2012 Received in revised form 20 July 2012 Accepted 30 July 2012 Available online 7 August 2012 Keywords: Porcine reproductive and respiratory syndrome virus (PRRSV) Genomic sequence Virulence Pathogenicity

a b s t r a c t Porcine reproductive and respiratory syndrome virus (PRRSV) is a ubiquitous and costly virus that exhibits substantial sequence and virulence disparity among diverse isolates. In this study, we compared the whole genomic sequence and virulence of 4 Type 2 PRRSV isolates. Among the 4 isolates, SDSU73, MN184, and NADC30 were all clearly more virulent than NADC31, and among the 3 more virulent isolates, there were subtle differences based on viral replication, lung lesions, lymphadenopathy, febrile response, decreased weight gains, and cytokine responses in the lung. Lesions consistent with bacterial bronchopneumonia were present to varying degrees in pigs infected with PRRSV, and bacteria typically associated with the porcine respiratory disease complex were isolated from the lung of these pigs. Genomic sequence evaluation indicates that SDSU73 is most similar to the nucleotide sequence of JA142, the parental strain of Ingelvac® PRRS ATP, while the nucleotide sequences of NADC30 and NADC31 are more similar to strain MN184. Both the NADC30 and NADC31 isolates of PRRSV, isolated in 2008, maintain the nonstructural protein 2 (nsp2) deletion seen in MN184 that was isolated in 2001, but NADC31 has two additional 15 and 36 nucleotide deletions, and these strains are 8–14% different on a nucleotide basis from the MN184 strain. These results indicate that newer U.S. Type 2 strains still exhibit variability in sequence and pathogenicity and although PRRSV strains appear to be reducing the size of the nsp2 over time, this does not necessarily mean that the strain is more virulent. Published by Elsevier B.V.

1. Introduction Porcine reproductive and respiratory syndrome virus (PRRSV) is a widely disseminated and economically important virus of swine that was first recognized as acute outbreaks of reproductive failure in the late 1980s. PRRSV is a linear positive-sense, single-stranded RNA virus that is a member of the genus Arterivirus in the family Arteriviridae and order Nidovirales (Cavanagh, 1997; Conzelmann et al., 1993; Meulenberg et al., 1993). One characteristic of this virus family is its ability to undergo high frequency mutation and viral recombination, which leads to remarkable evolution and extraordinarily diverse viruses (Hanada et al., 2004). PRRSV is divided into Type 1 (European-like; prototype strain Lelystad) and Type 2 (North American-like; prototype strain VR-2332) genotypes that vary in nucleotide sequence by approximately 40% (Nelsen et al., 1999).

∗ Corresponding author at: National Animal Disease Center, USDA, Agricultural Research Service, 1920 Dayton Avenue, P.O. Box 70, Ames, IA 50010, United States. Tel.: +1 515 337 7221; fax: +1 515 337 7428. E-mail address: [email protected] (S.L. Brockmeier). 0168-1702/$ – see front matter Published by Elsevier B.V. http://dx.doi.org/10.1016/j.virusres.2012.07.030

Within each genotype, nucleotide variation of over 20% is seen between isolates (Faaberg et al., 2006a). As a result, considerable genetic, antigenic and virulence differences exist among PRRSV isolates and depending on strain, dose and immune status, some farms may be subclinically infected with PRRSV while others experience severe reproductive and/or respiratory disease. Because of this heterogeneity, vaccines provide incomplete protection against heterologous viruses and thus it is essential to identify virulence determinants of PRRSV for rationale vaccine design. Unfortunately, identification of specific genomic determinants of virulence has been elusive and is almost certainly multifaceted. The PRRSV genome is 15.0–15.5 kilobases (kb), is polyadenylated, and is comprised of a 5 -untranslated region (UTR), at least 9 open reading frames (ORFs) that encode specific viral proteins, and a 3 -UTR (Meulenberg et al., 1993; Wu et al., 2001). Approximately 75% of the genome constitutes ORFs 1a and, through a ribosomal frameshift, 1ab that encode a replicase polyprotein. The polyprotein is processed by self-encoded proteases responsible for autocatalysis into at least 13 nonstructural proteins (nsps). The 3 end of the genome (about 3.5 kb) contains ORFs 2a and 2b through ORF7 that encode major and minor viral structural proteins. Viral

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transcription is characterized by the formation of a 3 -co-terminal nested set of 6 subgenomic messenger RNAs, each with a common 5 leader sequence (5 -UTR). Some comparative genomic work attempting to identify determinants of virulence has focused on the nsp2 protein of PRRSV. Nsp2 is a multidomain protein that is the most divergent protein between PRRSV Types 1 and 2 (Allende et al., 1999; Nelsen et al., 1999). Deletions in nsp2 were described in the highly virulent isolates coming out of Asia associated with the porcine high fever disease outbreaks. However, data also indicate that high mutation rates in nsp2 and tolerance to insertions and deletions occur with no correlation to virulence, suggesting that nsp2 mutations and deletions are not solely responsible for virulence differences (Allende et al., 1999; Fang et al., 2004, 2006; Gao et al., 2004; Han et al., 2006; Kim et al., 2007; Nelsen et al., 1999; Ran et al., 2008; Ropp et al., 2004; Shen et al., 2000). In this study, we compare the genomic sequence and virulence of four Type 2 PRRSV isolates, two pathogenic isolates that were associated with the emergence of novel and/or atypical North American PRRSV outbreaks in the late 1990s to early 2000s (Han et al., 2006; Mengeling et al., 1998) and two more contemporary United States isolates from herds experiencing respiratory problems. The goal of the research was to assess the virulence properties of more current Type 2 PRRSV alongside of known established pathogenic isolates, and to deduce and analyze the full genomic sequence of three additional U.S. strains of PRRSV. 2. Materials and methods 2.1. PRRSV isolates PRRSV MN184 was isolated in 2001 when novel, virulent PRRS viruses having restriction fragment length polymorphism (RFLP) patterns of ORF5 known as 1-8-4 were being isolated in Minnesota (Han et al., 2006). The MN184 virus had been passaged twice in MARC-145 cells. PRRSV SDSU73 was isolated in 1996 from a herd with a high prevalence of abortions, increased sow mortality, and severe illness among nursery age pigs described as atypical PRRS at the time (Mengeling et al., 1998). The SDSU73 virus had been passaged 4 times in MARC-145 cells. NADC30 and NADC31 isolates of PRRSV were isolated in 2008 from Iowa herds experiencing outbreaks of respiratory disease. These viruses were originally isolated on MARC-145 cells and passaged twice more in these cells. Swine influenza virus was isolated in conjunction with the NADC31 isolate of PRRSV.

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median cell culture infectious doses (CCID50 ) of PRRSV intranasally. Rectal temperatures were taken from day −3 to day 13 relative to PRRSV challenge. Pigs were weighed on days 0, 7, and 14 and blood samples were collected on days 0, 1–5, 7, 10, and 14 relative to PRRSV challenge. All pigs from the 4 PRRSV infected groups and 4 of the mock-infected pigs were euthanized 14 days after challenge with PRRSV. At necropsy, the same tracheobronchial lymph node was identified for each of the pigs, dissected out and weighed. The lymph node:body weight ratio was calculated by dividing the lymph node weight in grams by the total body weight of the pig in kilograms. Lung lavage for virus and bacterial isolation and cytokine analysis was obtained by instilling 50 ml of minimal essential cell culture medium into the lung through the trachea near the bifurcation and aspirating as much as possible back out with a pipette. During macroscopic examination, an estimate of the percentage of lung with both viral interstitial pneumonia, characterized by multifocal to diffuse, tan to red mottled areas with irregular borders, and bacterial bronchopneumonia, characterized by plum colored hepatization with clearly demarcated borders, was assigned on the basis of the percentage of each lung lobe affected and the percentage of total lung volume represented by each lobe. Percentage of total lung volume of each lobe was estimated as 10% for each cranial lobe, 10% for each middle lobe, 27.5% for each caudal lobe, and 5% for the intermediate lobe. 2.4. PRRSV isolation and titration Virus isolation was performed by adding 50 ␮l of serum or lung lavage to a monolayer of MARC-145 cells in 1 well of a 24 well plate. Each well was examined for cytopathic effect (CPE) and assessed as positive or negative daily for one week of culture. Titration was completed by preparing 10-fold dilutions of each positive sample and adding 50 ␮l of each dilution to 4 wells of a monolayer of MARC145 cells in a 96 well plate. The Reed and Muench method was used to calculate the 50% end point titer. Results were converted to log10 values for graphing and statistical analysis. 2.5. Serology Serum samples were tested from study days 0, 7, 10 and 14 for antibody response with the PRRS 2XR enzyme-linked immunosorbent assay (HerdChek ELISA; IDEXX Laboratories). A sample was considered positive for antibodies to PRRSV if the sample-topositive (S/P) ratio was equal to or greater than 0.4.

2.2. PRRSV genomic sequencing SDSU73, NADC30 and NADC31 full-length genomes were sequenced using 454 technology as described, except for the 5 and 3 ends and a few small gaps which were completed using standard methods (Guo et al., 2011). The complete genome sequences of SDSU73 (JN654458), NADC30 (JN654459) and NADC31 (JN660150) have been deposited in GenBank. The nucleotide sequences were deduced and analyzed using various programs included in Geneious Pro Version 5.4.6 (Biomatters Ltd., Auckland, New Zealand). Alignments were completed using PRRSV Type 2 reference strains VR-2332 (U87392), JA142 (AY424271) and MN184C (EF488739). 2.3. Pig experimental design Forty-nine, 5-week-old pigs from a herd negative for PRRSV antibody were divided into 4 groups of 10 pigs, each given one of the PRRSV strains, and 1 group of 9 mock-infected pigs challenged with medium from non-infected cell culture. On day 0 of the experiment, pigs were challenged with 2 ml containing 104

2.6. Multiplex cytokine assay Approximately 5 ml of lung lavage was centrifuged at 300 × g for 10 min at 4 ◦ C to pellet cellular debris. The cell-free lung lavage was used to assay for levels of IL-1␤, IL-8, IL-6, TNF-␣, IL-2, IL-4, IL12p70, IFN-␥, and IL-10 by SearchLight multiplex ELISA performed according to the manufacturer’s recommendations (Aushon Biosystems, Billerica, MA). The average of duplicate samples for each sample was used for statistical analysis. 2.7. Bacterial isolation and identification Bacterial culture was performed by plating 100 ␮l of lung lavage on both a Casman’s agar plate with added NAD and a 5% sheep blood agar plate and incubating for 48 h at 37 ◦ C. Bacterial identification was performed by 16S rRNA-specific PCR and DNA sequencing. The presence of Mycoplasma hyopneumoniae DNA in lung lavage was determined by real-time PCR as previously described (Strait et al., 2008).

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2.8. 16S rRNA-specific PCR and DNA sequencing Bacterial whole-cell lysates used as templates for 16S PCR were prepared by suspending a colony, ∼2 mm in diameter or the equivalent, in 50 ␮l of sterile water. The mixture was boiled for 10 min, placed on ice until chilled, and centrifuged at 16,000 × g for 1 min to pellet cell debris and stored at −20 ◦ C. Supernatant (5 ␮l) was used as the template in each PCR. The forward primer (5 -AGAGTTTGATCCTGGCTCAG-3 ), designated univ16S-3, is homologous to a highly conserved sequence from the 5 end of the 16S rRNA gene and the reverse primer (5 -GCGGCTGCTGGCACG3 ), designated univ16S-4, is homologous to a highly conserved sequence between the third and fourth variable regions of the 16S rRNA gene. This previously described primer set generates an amplicon of approximately 520 bp (Register and Yersin, 2005). Reactions were carried out in a volume of 50 ␮l and contained 2 U AmpliTaq polymerase (Applied Biosystems, Foster City, CA), 5 ␮l 10× buffer II (100 mM Tris–HCl, pH 8.3, 500 mM KCl), 5 ␮l dimethyl sulfoxide (DMSO), 1.5 mM MgCl2 , 0.5 ␮M primers, and 200 ␮M deoxynucleoside triphosphates. An initial denaturation step of 5 min at 95 ◦ C was followed by 35 cycles of 20 s at 94 ◦ C, 30 s at 55 ◦ C, and 3 min at 72 ◦ C, with a final extension step of 10 min at 72 ◦ C. Ten microliters of each PCR reaction volume was analyzed by agarose gel electrophoresis and PCR products were purified with spin columns (Promega, Madison, WI) and sequenced directly by fluorescence-based cycle sequencing with AmpliTaq and BigDye Terminators on an ABI 377 sequencer at the National Animal Disease Center Genomics Unit. Sequences were analyzed using Geneious 5.0 software (Biomatters Ltd., Auckland, New Zealand). 2.9. Statistics Rectal temperature and serum viral titers were analyzed using a mixed linear model for repeated measures (SAS 9.1 for Windows, SAS Institute Inc.). Linear combinations of the least squares sample estimates were used in a priori contrasts after testing for either a significant (P < 0.05) treatment effect or an interaction effect between time-point and experimental treatment. Comparisons were made between treatment groups at each time-point using a 5% level of significance (P < 0.05) to assess statistical differences. The mean percentage of macroscopic lung lesions, lymph node:body weight ratios, cytokine levels in the lung lavage, and viral titers in the lung lavage were analyzed using GraphPad Prism’s one way ANOVA applying Tukey’s post test and a significance level of P < 0.05. 3. Results 3.1. PRRSV genomic sequences and alignments In order to gain a better genetic understanding of Type 2 strain SDSU73, a virulent virus isolated in 1996 that has been used frequently in pathogenesis studies (Brockmeier et al., 2009; Faaberg et al., 2006b; Kim and Yoon, 2008; Loving et al., 2008; Opriessnig et al., 2007), and two recent isolates associated with severe respiratory disease, the full-length sequences of Type 2 strains SDSU73, NADC30 and NADC31 were obtained using 454 methodology (Guo et al., 2011). The genome of strain SDSU73 was found to be 15,412 bases in length exclusive of the poly A tail. The genomes of strains NADC30, 15,020 bases, and NADC31, 14,968 bases, were considerably shorter. Each of these strains was found to encode a novel Type 2 genome that had 82–88% nucleotide identity with each other. SDSU73, NADC30 and NADC31 were aligned to VR-2332 (15,411 bases), JA142 (15,412 bases) and MN184 (15,019 bases), representing three major Type 2 subgenotype representatives present

VR-2332 JA142 SDSU73 NADC30 MN184 NADC31 Fig. 1. Phylogenetic analysis of the complete genomes of main Type 2 genotypes detected in the United States with SDSU73, NADC30 and NADC31. Analysis of the full-length alignment was completed using the Geneious Tree Builder application, Jukes-Cantor distance model and Neighbor-Joining tree build method with 500 bootstrapping replicates.

in the US. VR-2332 differs from JA142 by 9.9% pairwise identity on the nucleotide basis and from MN184 by 16.3%, and JA142 and MN184 differ by 16.6%. Phylogenetic analysis of the alignment was derived (Fig. 1). SDSU73 was most similar to the nucleotide sequence of JA142 (95.9% pairwise identity), the parental strain of Ingelvac® PRRS ATP, while the nucleotide sequences of NADC30 (86.6% identity) and NADC31 (92.4% identity) were more similar to strain MN184. Independent full-length BLAST® analysis (United States National Center for Biotechnology Information) confirmed these results (data not shown). To understand the areas of the genomes that contained the most variability when compared to the genetically closest reference strain, the completed alignments were broken down into individual segments (Table 1). As noted above, strain SDSU73 possessed 95.9% pairwise nucleotide similarity to strain JA142. The most variable regions (<95% pairwise nucleotide identity) between the genomes of these two strains were found to lie within the nsp7␣ and nsp7␤ regions of ORF1a, ORF5 and the 3 -UTR. However, SDSU73 proteins nsp1␤, nsp2, nsp7␤, GP2-5, and the amino acids coded for the newly identified ORF5a (Johnson et al., 2011) were shown to diverge more than 5% in pairwise amino acid identity from JA142. The SDSU73 genomic regions of lesser identity to strain JA142 were therefore spread throughout the genome, and the newly sequenced virulent genome was not a result of immediately detectable viral recombination (data not shown). Furthermore, there were no insertions or deletions when comparing JA142 and SDSU73 genomes. The genome of strain NADC30, when compared to strain MN184, was more divergent than was detected between SDSU73 and JA142 (Table 1). All but a few segments had more than a 5% difference on the nucleotide level, with several areas revealing more than 15% nucleotide divergence including the ORF1a regions of nsp3–5 and 7␣, and ORF3. On amino acid comparison between strains NADC30 and MN184, only GP3 revealed less than 85% pairwise identity, but several other proteins had less than 90% identity including nsp1␤, nsp2, nsp5, nsp7␤, GP2, GP3 and GP5. However, when NADC31 was compared to MN184, the genome was of a greater percent nucleotide pairwise identity than NADC30 in all regions of ORF1ab except nsp10, several of the structural protein ORFs and the 3 -UTR. Finally, when surveying the percent pairwise amino acid identity, all proteins were of greater identity to strain MN184 as compared to NADC30, except four that were found to be only slightly less related. Again, no detectable viral recombination with MN184 was observed for NADC30 and NADC31, and the nucleotide and protein differences were spread throughout the genome. However, even though both newly obtained genomes were quite divergent from

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Table 1 Variability in various genomic regions when compared to the genetically closest reference strain of PRRSV. Genomic region

Length (nt/aa)

5 -UTR Nsp1a Nsp1b Nsp2 Nsp3 Nsp4 Nsp5 Nsp6 Nsp7a Nsp7b Nsp8 Nsp9 Nsp10 Nsp11 Nsp12 GP2 E GP3 GP4 ORF5a GP5 M N 3 -UTR

192/– 540/180 1827/609 3144–3588/1048–1196 690/230 612/204 510/170 48/16 447/149 330/110 138/45 2055/685 1323/441 669/223 465/154 771/256 222/73 765/254 537/178 141–156/46–51 603/200 525/174 372/123 151/–

Pairwise % identity (nt/aa) SDSU + JA142

NADC30 + MN184

NADC31 + MN184

96.3 99.1/99.4 95.2/94.6 95.7/94.0 98.0/98.7 97.7/99.0 98.2/99.4 100.0 93.1/97.3 93.3/94.5 95.6/96.4 96.8/99.1 95.6/98.6 95.1/97.8 97.8/99.4 95.6/93.4 95.0/97.3 94.6/93.3 95.2/93.8 89.4/94.1 92.7/91.0 97.1/97.1 93.3/97.6 93.4

97.4 91.3/95.6 88.3/87.2 86.9/85.9 84.1/90.9 83.7/92.2 84.7/88.2 100.0 84.3/91.3 90.6/89.1 97.8/94.9 92.9/97.1 95.2/99.1 87.0/93.3 85.9/91.6 86.9/89.1 94.1/94.5 84.1/83.5 91.8/89.9 95.7/91.3 90.9/91.6 90.3/94.8 91.7/92.7 96.7

95.3 93.7/96.1 91.5/91.1 98.9/88.9 95.8/97.5 95.1/98.0 91.8/89.4 100.0 89.3/91.9 93.3/92.7 93.5/93.5 95.4/98.1 94.3/98.9 91.6/95.5 93.1/96.8 94.9/87.1 92.8/95.9 94.9/95.3 88.6/90.4 88.7/91.3 87.2/96.8 89.9/95.4 91.9/91.9 92.7

MN184, NADC30 directly mimicked the nsp2 deletion signature of 333, 3, and 57 bases when compared to the prototype strain VR2332. NADC31 maintained the same 333 and 57 base deletions, but extended the middle deletion to 18 bases and possessed an additional 36 base deletion analogous to nucleotides 3083–3118 of VR-2332. NADC31 thus represents the smallest genome of any PRRSV field isolate sequenced to date. The contribution of nsp2, the most variable nucleotide and protein region of the genome having a nucleotide identity of 31.1% between all Type 2 PRRSV genomes and only 9.3% between all fulllength Type 1 and 2 sequences, to viral pathogenesis is not clear but has been an area of acute interest. The global nsp2 protein alignment of the five genetically similar strains discussed above is shown in Fig. 2A. SDSU73 differs from its genotype index strain primarily in the two hypervariable regions of nsp2 protein. NADC30 possessed amino acid changes from those of strain MN184 spread throughout the protein, and NADC31 revealed mostly amino acid changes from those of MN184 in the hypervariable regions. While the nsp2 sequence of both NADC30 and NADC31 diverge significantly from MN184, they each deviate differently, having the identical amino acid difference at only 35 positions when compared to MN184 (∼3%). A detailed alignment of the PLP2 protease signature showed that critical residues for protease activity as defined previously for protease cleavage (catalytic C54 and H123 ; as well as C110 , W124 , C141 , and C145 ) at or about VR-2332 amino acid 1196 G|G|G were maintained as were residues important for cleavage in trans (Han et al., 2009; Snijder et al., 1995) (Fig. 2B). Cysteine54 has also been linked with H218 and D220 as the catalytic triad signature of the ovarian tumor protease (OTU) family of deubiquitinases (DUBs), which cleaves after G|G dipeptides (Capodagli et al., 2011; Komander, 2010; Komander et al., 2009). The OTU catalytic triad is also conserved between the five Type 2 strains. However, several amino acid differences are seen in the PLP2 protease signature sequence outside of the conserved catalytic residues mentioned, and the possible contribution of these nsp2 alterations to PRRSV virulence are as yet unknown. In order to examine the deletions seen in NADC31 alongside of all viruses containing deletions or insertions in the second

hypervariable domain of nsp2 relative to strains VR-2332 (Type 2), JA142 (Type 2) which includes the Asian highly pathogenic strains, MN184 (Type 2) or Lelystad (Type 1), all nsp2 nucleotide sequences deposited in GenBank were aligned. After translation, the amino acid sequences were aligned and clustered by similarity. From the protein alignment, sequences that did not contain deletions or insertions from their respective index strain stated above were eliminated. The resulting display (Fig. 2C) revealed there are very few areas of similarity between all PRRSV deletion mutants (dark gray) and that deletions of up to 403 amino acids in the hypervariable region can be made without loss of replication in vivo, although most deletions found in natural isolates are less than or equal to 111 amino acids. We also examined ORF 5 properties more closely because the restriction fragment length polymorphisms (RFLPs) for this region is a common method of categorizing PRRSV field isolates. The ORF 5 RFLPs were calculated to be 1-4-4 for SDSU73 and NADC30 strains, and 1-24-2 for strain NADC31 (Larochelle et al., 2003; Wesley et al., 1998). The GP5 amino acid alignment revealed that these 3 novel strains share the predicted N-glycosylation sites at N45 and N51, as would be predicted for the identified primary neutralizing site on the virus (Plagemann et al., 2002), but the variability in the surrounding HV-1 and HV-2 domains is considerable (Fig. 3). The predicted N-glycans in HV-1, thought to be signatures for host Bcell response evasion, vary from one (NADC30) to three (NADC31) sugar moieties. 3.2. PRRSV replication in vivo PRRSV was isolated from the serum of all pigs challenged with virus but from none of the mock-infected control pigs. On day 1 post exposure, PRRSV was detected in the sera of 8/10 pigs challenged with NADC30 as compared to only 2/10 pigs challenged with SDSU73 or MN184. By day 2, PRRSV was detected in the serum of the majority of the pigs in these 3 groups. PRRSV was not isolated from the serum of any pig challenged with NADC31 until day 3 post exposure, and then in only 4/10 pigs, and not isolated from the rest of the pigs in this group until day 7 or later. Viral titers in the serum were highest (except for day 1) and similar for MN184 and

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Fig. 2. Nsp2 protein alignments. (A) The top schematic represents the organization of the nsp2 protein of PRRSV showing the relative positioning of the PLP2 enzyme domain in between the two hypervariable regions (HV1 and HV2) of unknown function. The putative transmembrane domains (TM; VR-2332 aa875–897, 910–929, 964–980 and 988–1008) are also shown. Below are the two sets of related strains analyzed in this report; the index strain sequences shown as horizontal black bars and the newly sequenced viruses by gray bars. The amino acid differences from the related index isolate are represented as vertical black lines within the gray bars. (B) The sequence of the proposed core PLP2 protease domain. Critical residues for PRRSV strain VR-2332 PLP2 cleavage at the nsp2/3 junction, including the catalytic cysteine (C54 ) and histidine (H123 ) as well as other cysteine and trytophan residues (C110 , W124 , C141 , C145 ) are shown as black bars above the residues. Those residues shown to be critical only in VR-2332 trans-cleavage are represented by gray bars (G55 , D84 , W85 , and D88 ) (Han et al., 2009). The residues recognized as being critical for OTU activity include C54 , H218 and D220 ; H218 and D220 are shown as an open bar (Capodagli et al., 2011; Komander, 2010). (C) Schematic representing all replicating Type 2 PRRSV strains that show deletions or insertions in nsp2 HV-2 domain. Sequences were aligned using greyscale to show amino acid relative similarity between PRRSV strains. Similarity score (80–100% – dark gray, 60–80% – medium gray, and <60% – light gray) was based on the Blosum62 matrix.

SDSU73 throughout the experiment (Fig. 4A). NADC30 was in general detected earlier, but beyond day 1, viral titers fell below those for SDSU73 and MN184 and above those for NADC31 until day 10 post exposure. NADC31 viral titers were the lowest but approximated those for the other groups at the end of the experiment as viral titers for this group were still rising and those for the other PRRSV infected groups were steady or declining. PRRSV was isolated from the lung lavage on day 14 postexposure of all pigs challenged with virus, but not from any of

the mock-infected control pigs. Viral titers in the lung lavage were highest for pigs infected with SDSU73 and MN184, and these were statistically greater than the viral titers for pigs infected with the NADC31 isolate of PRRSV (Fig. 4B). Pigs infected with the NADC30 isolate had viral titers between those of pigs infected with either SDSU73 or MN184 and those of pigs infected with NADC31. Pigs in the groups infected with SDSU73, MN184, and NADC30 behaved similarly with regard to seroconversion to PRRSV (Fig. 4C). A few pigs had seroconverted by day 7, the majority by day 10,

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Fig. 3. GP5 amino acid alignment. A hatched box represents the predicted signal sequence and the transmembrane regions are shown by the solid boxes, predicted by InterProScan. The two hypervariable regions, HV-1 and HV-2, surround the more conserved N-glycan residues (夽) and the completely conserved cysteine residue (↓) that interacts with a conserved cysteine residue on the membrane protein via a disulfide bond (Faaberg et al., 1995; Snijder et al., 2003).

and all but 1 pig in the MN184 infected group seroconverted by day 14 of the experiment. Of the pigs in the group infected with NADC31, only 1 had seroconverted by day 10 and 6 by day 14 of the experiment, reflecting the delay in viral detection in these pigs. None of the mock-infected control pigs seroconverted to PRRSV.

Weight gain is shown in Fig. 5B. Weight gain was least for the SDSU73 infected group followed by the MN184 infected group that had average daily gains of 0.152 kg and 0.238 kg, respectively. There was little difference in weight gain among the NADC30, NADC31, and mock-infected control groups, which had average daily gains of 0.298 kg, 0.321 kg, and 0.323 kg, respectively.

3.3. Febrile response and weight gain

3.4. Pneumonia and lymphadenopathy

Fig. 5A illustrates the febrile response of the pigs. Rectal temperatures rose earliest in pigs infected with NADC30 with an early peak at 2 days post infection. Pigs infected with SDSU73 had an early febrile peak at day 2 and a second peak at day 8 post-infection. Temperatures for the MN184 infected pigs were slower to rise and peaked at day 8. The mean rectal temperature for mock-infected pigs stayed below 40 ◦ C for the duration of the experiment. The NADC31 infected group had mean rectal temperatures that rose above 40 ◦ C on days 3, 7, and 8 post challenge, while those for the NADC30 group were above 40 ◦ C on days 1–3, 6, and 8–11. The febrile response for the SDSU73 and MN184 infected groups were highest overall and above 40 ◦ C on days 2–11 and 6–11, respectively. Mean rectal temperature for the NADC30, MN184, and SDSU73 groups was overall statistically greater than that for mock-infected controls. Mean rectal temperature for the MN184 and SDSU73 groups was also overall statistically greater than that for the NADC31 infected group.

All pigs infected with PRRSV had lesions consistent with interstitial pneumonia, characterized by multifocal to diffuse, tan to red mottled areas with irregular borders that tended to be of greatest severity in the cranioventral portions of the lungs. Some of the PRRSV infected pigs also had lesions consistent with bacterial bronchopneumonia characterized by plum colored hepatization with clearly demarcated borders. The lungs from the mock-infected pigs appeared macroscopically normal. Based on an estimate of lung involvement, viral interstitial lung lesions were most extensive in pigs infected with SDSU73 and MN184 affecting an average of 82–71% of the lung, respectively (Fig. 6A). Viral interstitial pneumonia was relatively mild in pigs infected with the NADC31 isolate of PRRSV, while pigs infected with the NADC30 isolate were moderately affected. Viral pneumonia was significantly greater for pigs infected with the SDSU73, NADC30, and MN184 isolates of PRRSV as compared to pigs infected with the NADC31 isolate and mockinfected pigs. Lesions consistent with bacterial pneumonia (well

Fig. 4. PRRSV replication in vivo and seroconversion. Levels of PRRSV detected by virus titration on MARC-145 cells in the serum at various days after challenge (A) or lung lavage 14 days after challenge (B) with SDSU73, MN184, NADC30, or NADC31 strains of PRRSV. Antibody response as measured by ELISA (C). Samples are considered positive for antibodies to PRRSV if the sample-to-positive (S/P) ratio is equal to or greater than 0.4. The data are expressed as the mean ± SEM of 10 pigs. No PRRSV was detected from any of the samples collected from pigs in the Mock infected group. Different letter superscripts denote significant differences at P < 0.05.

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Fig. 5. Febrile response and weight gains after PRRSV challenge. Rectal temperature (A) and body weight (B) in groups of pigs after challenge with SDSU73, MN184, NADC30, or NADC31 strains of PRRSV or mock challenged with medium from non-infected cell culture. Data are expressed as the mean of 10 pigs in the PRRSV infected groups and 9 pigs in the mock infected group.

demarcated tan to plum colored consolidation with a cranial ventral distribution) were present in 3/10 pigs infected with SDSU73 (affecting an average of 21% of the lung in pigs with lesions), 5/10 pigs infected with MN184 (affecting an average of 15% of the lung in pigs with lesions), 3/10 pigs infected with NADC30 (affecting an average of 8.7% of the lung in pigs with lesions), and 1/10 pigs infected with NADC31 (affecting 6% of the lung). Mild to moderate lymphadenopathy was noted in the pigs infected with PRRSV. Lymph node:body weight ratios were significantly greater for pigs infected with the SDSU73, NADC30, and MN184 isolates of PRRSV as compared to pigs infected with the NADC31 isolate and mock-infected pigs (Fig. 6B). Only slight lymph node enlargement was seen in the NADC31 infected pigs, and the lymph node:body weight ratios were not significantly different from those of mock-infected controls. 3.5. Bacterial isolations from lung lavage >Bordetella bronchiseptica was isolated from the lung lavage of all pigs including the controls. Haemophilus parasuis was isolated from 5/10 pigs infected with SDSU73, from 6/10 pigs infected with MN184, from 8/10 pigs infected with NADC30, from 5/10 pigs

infected with NADC31, and 3/4 control pigs. Streptococcus suis and Pasteurella multocida were only isolated from pigs infected with PRRSV; S. suis from 1/10 pigs in groups infected with SDSU73, NADC30, and NADC31 and from 4/10 pigs infected with MN184; and P. multocida from 1/10 pigs infected with SDSU73 and 4/10 pigs infected with MN184. P. multocida was always isolated in association with B. brocnshiseptica and S. suis. Of the 12 pigs with macroscopic evidence of bacterial pneumonia, B. bronchiseptica, S. suis and P. multocida were isolated from 5, B. bronchiseptica, H. parasuis and S. suis were isolated from 2, and B. bronchiseptica and H. parasuis were isolated from 5. Co-isolation of B. bronchiseptica, S. suis and P. multocida was associated with the most severe lesions affecting an average of 23% of the lung, compared to 8.4% and 6.5% for lungs with bacterial pneumonia lesions where B. bronchiseptica and H. parasuis, or B. bronchiseptica, H. parasuis, and S. suis were isolated. No M. hyopneumoniae DNA was detected in the lung lavage of any of the pigs via PCR. 3.6. Cytokine levels in the lung lavage Cytokine levels in the lung lavage were evaluated by multiplex ELISA and results showed an increased production of several

Fig. 6. Pneumonia and lymphadenopathy after PRRSV challenge. The mean estimated percentage of lung lesion (A) and the lymph node:body weight ratio (lymph node weight in grams divided by body weight in kilograms) (B) of pigs 14 days after challenge with SDSU73, MN184, NADC30, or NADC31 strains of PRRSV or mock challenged with medium from non-infected cell culture. Data are expressed as the mean ± SEM of 10 pigs in the PRRSV infected groups and 4 pigs in the mock infected group. Different letter superscripts denote significant differences at P < 0.05.

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Fig. 7. Cytokines in the lung lavage after PRRSV challenge. Cytokine levels in the lung lavage 14 days after challenge with SDSU73, MN184, NADC30, or NADC31 strains of PRRSV or mock challenged with medium from non-infected cell culture. Data are expressed as the mean ± SEM of 10 pigs in the PRRSV infected groups and 4 pigs in the mock infected group. Different letter superscripts denote significant differences at P < 0.05.

cytokines in pigs infected with PRRSV. In general, increases in the amounts of IL-1␤, TNF-␣, IL-8, IFN-␥, and IL-10 were detected in PRRSV infected pigs compared to non-infected controls (Fig. 7). However, elevations of these cytokines were mild in the group of pigs infected with the NADC31 isolate of PRRSV. Overall, the levels of these cytokines were quite similar between the SDSU73 and MN184 groups, with both these groups having statistically higher mean concentrations of IL-8 and IFN-␥ than NADC31 pigs while NADC30 pigs fell in between, but not statistically different from the other PRRSV infected groups. Pigs infected with the NADC30 isolate of PRRSV had statistically higher mean concentrations of TNF-␣ and IL-10 than NADC31 pigs with SDSU73 and MN184 pigs falling in between, but not statistically different from the other PRRSV infected groups. 4. Discussion The considerable genetic, antigenic and virulence diversity that exists among PRRSV isolates makes surveillance of recently circulating strains important. In the present study we compared the genomic sequence and virulence of 4 Type 2 PRRSV isolates from the United States to determine the pathogenicity of current PRRSV field isolates in the U.S. and to separately determine how the PRRSV genome is evolving. In general, MN184 and SDSU73 behaved very similarly in vivo, growing to similar viral titers and inducing similar lung lesions and cytokine levels, although SDSU73 induced an earlier febrile response and increased lymphadenopathy. NADC31 was clearly the least virulent and NADC30 typically fell somewhere in between. However, pigs infected with NADC30 were the quickest to develop viremia and this corresponded to an early febrile response that was as high as any of the isolates for the first 4 days post challenge, and in many of the parameters measured NADC30 was not statistically different than either MN184 or SDSU73. Therefore, there were subtle differences among SDSU73, MN184 and NADC30, but they all were more virulent than NADC31. Both NADC30 and NADC31 were isolated from herds exhibiting significant respiratory disease, the fact that swine influenza was isolated in conjunction with NADC31 may explain the similar severity in clinical disease in these herds despite the variability in virulence between the two isolates. We determined the full-length nucleotide sequence of three of the strains of PRRSV in this study using pyrosequencing methods (Guo et al., 2011), while the fourth strain (MN184) had already been sequenced. SDSU73 was most similar to index strain JA142, and had no deletions in nsp2. Although more similar to the footprint of strain MN184, NADC30 and NADC31 were nevertheless quite distinct from each other, having only 35 amino acid changes in common when compared to MN184. Of significance in analyzing

the genetic data, there is no single specific region that is altered. SDSU73 is quite distinct from JA142 throughout the genome, even though it was isolated in the same year and causes similar clinical disease. Infection with NADC30 results in a similar clinical outcome as MN184, has the same genetic footprint as MN184, but differs in sequence by more than 10% throughout the genome, while NADC31 replicates poorly in vivo, yet has the same genetic footprint as MN184 and is genetically more similar (92.4%) to MN184 than NADC30. PRRSV strains appear to be reducing the size of nsp2 over time, but this fact does not necessarily equate to the strain being more virulent. Recently, strains of highly pathogenic PRRSV have emerged in China and elsewhere in Asia with hallmark deletions in nsp 2, originally thought to potentially be involved in the increased virulence seen with these strains (Tian et al., 2007). However, further work revealed that although the nsp2 deletions were a key characteristic of these viruses, it did not directly relate to virulence (Zhou et al., 2009). In addition to not finding any specific region that correlates to virulence, there also appears to be no single specific region that is altered in classical attenuation methods. When the sequence of two attenuated vaccine strains of PRRSV were compared to their parent viruses, 41 nucleotides were altered in attenuation of VR-2332 to RespPRRS® MLV and 58 nucleotides were altered in attenuation of JA142 to Ingelvac® ATP, but no common sites were detected (An et al., 2011; Yuan et al., 2001). The conclusion is that identifying virulence determinants from genetic comparisons is complex and attenuation/virulence is almost certainly strain specific. Upon further examination, variability in amino acid sequence was also seen in the nsp2 PLP2 protease that has been identified as a member of OTU family of deubiquitinating enzymes. The PLP2 OTU protease of Type 1 PRRSV has been associated with the ability of nsp2 to inhibit NF-␬B activation and has been shown to deubiquitinate in vitro, but the critical residues for PRRSV cleavage after glycine residues have not been explored in this genotype (Sun et al., 2010). Using the Type 2 prototype strain VR-2332, it was biochemically determined that the catalytic C54 and H123 , as well as C110 , W124 , C141 , and C145 were responsible for cleavage at or near the signature sequence of G|G|G at amino acid 1196 (Han et al., 2009). However, when VR-2332 infected MARC-145 cells were examined, many nsp2 cleavage products were seen (Han et al., 2010). Since the catalytic triad of the OTU DUB in PRRSV nsp2 protease has been predicted to be C54 , H218 and D220 (Capodagli et al., 2011), and DUBs cleave after G|G dipeptides (Komander, 2010; Komander et al., 2009) of which there are several in PRRSV nsp2, whether or not these differing catalytic residues represent two distinct protease activities remains to be clarified. PRRSV has been shown to predispose to secondary bacterial infections, and the lung lesions seen in this experiment that were

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consistent with bacterial pneumonia are consistent with this supposition. B. bronchiseptica and H. parasuis were found in the lung lavage of both non-infected controls and PRRSV infected pigs, but only PRRSV infected pigs had lesions consistent with bacterial pneumonia. PRRSV has been shown to induce bronchopneumonia with B. bronchiseptica in coinfected pigs (Brockmeier et al., 2000), and B. bronchiseptica has been shown to predispose to colonization with P. multocida, H. parasuis and S. suis (Brockmeier, 2004; Brockmeier et al., 2001; Vecht et al., 1989, 1992). In the current experiment, consolidation was most extensive in pigs from which B. bronchiseptica, S. suis and P. multocida were isolated. Previous studies have also shown that although PRRSV alone failed to induce bronchopneumonia with P. multocida, coinfection with PRRSV and B bronchiseptica interact to adversely affect respiratory tract defense mechanisms, leaving pigs especially vulnerable to infection with secondary agents such as P. multocida (Brockmeier et al., 2001). Overall, increased virulence was associated with higher titers of virus in vivo. However the temporal replication differences may be of more significance. NADC30 serum virus loads peaked early and then leveled off, whereas SDSU73 and MN184 had higher overall viral serum titers that peaked 5–7 days after challenge. NADC31 serum viral titers were slow to rise but were still increasing and similar to those of the other groups by day 14 post-challenge. Cytokines in the lung lavage in general were lowest for NADC31 and may be the reason for diminished clinical signs and lesions. SDSU73 and MN184 had very similar responses with IL-8 and IFN␥ levels most associated with the increased severity of disease seen. NADC30 tended to have the highest levels of TNF␣ and IL10. High levels of TNF␣ have been shown to induce production of IL-10 to control inflammatory responses, which may explain the higher levels of both these cytokines in NADC30 pigs (Cyktor and Turner, 2011). IL-10 has been shown to inhibit the production of other cytokines and could be one reason disease seen with NADC30 was somewhat diminished, but since IL-10 is also proposed to contribute to the immunosuppression and the weak and delayed immune response seen with PRRSV (Mateu and Diaz, 2008), it could also relate to the viruses evolving into less virulent but more persistent infection that may favor survival of the virus. It is important to remember the cytokine levels at day 14 reflect one time point and differences may reflect variations in viral replication both in maximal amounts and temporal diversity, as well as inherent differences in the viruses themselves. Additional experiments that follow the cytokine response elicited by PRRSV isolates exhibiting diversity in virulence and persistence are needed to specifically address these issues.

Acknowledgements The authors would like to thank Kim Driftmier, Gwen Nordholm, Sarah Anderson, and David Michael for their excellent technical assistance, and Jason Huegel, Brian Pottebaum, and Jason Crabtree for their excellent animal care. USDA is an equal opportunity provider and employer. Disclaimer: Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.

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